Pharmaceutical product comprising mite allergen extract(s) and a method for the manufacture thereof

ABSTRACT

The invention relates to a pharmaceutical product comprising an allergen extract or an allergoid thereof for the treatment and/or prevention of allergy and allergic asthma caused by house dust mites, which extract comprises at least one extract of mite bodies selected from the following groups a)-b): a) An extract of Der p mite bodies, and b) An extract of Der f mite bodies, and at least one extract of mite cultures selected from the following groups c)-g): c) An extract of Der p faecal particles, d) An extract of Der f faecal particles, e) An extract of Der f whole mite culture, f) An extract of an Der p whole mite culture, and g) a combination of extracts c) to f).

CROSS REFERENCE RELATED APPLICATIONS

This application is a continuation in part of PCT/EP2011/059210, filedJun. 3, 2011, which claims priority to EP10164879.8 filed Jun. 3, 2010and U.S. provisional application No. 61/351,164 filed Jun. 3, 2010, allof which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to a pharmaceutical product suitable for thetreatment and/or prevention of house dust mite allergy and allergicasthma caused by house dust mites comprising mite allergen and a methodfor the manufacture thereof.

BACKGROUND OF THE INVENTION

Allergy is a major health problem in countries with a Western lifestyle.Furthermore, the prevalence of allergic disease is increasing in thesecountries. Although allergy in general may not be considered alife-threatening disease, asthma annually causes a significant number ofdeaths. An exceptional prevalence in about 30% of teenagers conveys asubstantial loss in quality of life, working days and money, andwarrants a classification among major health problems in the Westernworld.

Clinical allergy manifestation and symptoms manifest themselves inseveral ways and may vary depending on the sensitized individual and theallergy inflicted. Common symptoms are edema, itching, redness andrunning of the eyes and nose (rhinitis and conjunctivitis) and symptomsof the upper and lower airway such as wheezing, coughing, shortness ofbreath, skin conditions like eczema, urticaria and itching. Othersymptoms like fatigue or insomnia are also experienced. Symptomatictreatment aims at reducing or affecting severity of the symptoms orreducing the need for other drugs given in parallel. Symptomatic drugsinclude antihistamines like H₁ and H₂ receptor antagonists, intranasaland systemic corticosteroids, non-steroid anti-inflammatory drugs, nasaldecongestants like adrenoceptor agonists. Treatment and relief of one ormore allergic symptoms and/or the reduction in the need for othermedication is a further object of this invention.

Allergen specific immunotherapy (also referred to as allergenimmunotherapy, allergy immunotherapy, specific allergy vaccination(SAV), hyposensitization or desensitization) is a causal treatment ofallergic disease. It interferes with basic immunological mechanismsresulting in persistent or long term improvement of the patients' immunestatus. Thus, the protective effect of specific allergy vaccinationextends beyond the treatment period in contrast to symptomatic drugtreatment. Some patients receiving the treatment are cured, and inaddition, most patients experience a relief in disease severity andsymptoms experienced, or at least an arrest in disease aggravation.Allergen immunotherapy also has preventive effects reducing the risk ofhay fever developing into asthma, and reducing the risk of developingnew sensitivities.

Conventional allergen immunotherapy is carried out using multipleadministration of allergen applied over an extended time period.

A long standing use of allergen immunotherapy is via the injection route(also called SCIT, SIT or subcutaneous immunotherapy). Commonly,allergen is administered via the subcutaneous route in incremental dosesuntil a maintenance dose is reached, but also treatment regimens withcluster administration or without up-dosing are used. Allergen may beadministered for a period up till 3 years or more before treatment iscompleted or ceased depending on the treatment schedule applied.

An alternative administration route is sublingual (SLIT) administrationwhich is more convenient for the patients as administration may becarried out at home.

The immunotherapy products for use in immunotherapy typically comprisethe allergen that causes the allergy in the patient or derivativesthereof as the active ingredient, which may be formulated in variousways according to the administration route of choice such as aqueousformulations, aluminum hydroxide suspensions or allergoids. The activeingredient may for example be an aqueous extract of pollen containingpollen allergens and/or aqueous extract of mites containing miteallergens.

The most important house dust mites belonging to the family ofPyroglyphidae include Euroglyphus maynei, Dermatophagoides pteronyssinusand Dermatophagoides farinae. These are found in most of the worldincluding Europe, USA, China and many other counties and allergy andallergic asthma caused by house dust mites is a common diseaseafflicting 25% of the population.

Some patients are sensitized to Euroglyphus maynei, Dermatophagoidespteronyssinus, some others to Dermatophagoides farinae and others toboth species.

Patients are normally sensitized (although to a variable degree) to thetwo major allergens in house dust mites, namely group 1 allergen and/orgroup 2 allergen.

House dust mites contain more than 20 different allergens. However, thetwo major allergens group 1 and group 2 are the most important allergensbecause of the frequency of patients sensitized to these two allergens,the amount of IgE produced in response to these allergens and thecontent thereof in natural extracts.

In allergen immunotherapy patients are treated with allergens that theyare sensitized to. The ability of allergens to elicit IgE mediatedimmunologically responses makes it important to control the allergencontent especially group 1 and group 2 allergens in the allergenextracts administered to allergic patients to avoid side effects.

The group 1 and 2 allergens from Der f and Der p are closely related butnot identical, see Allergens and Allergen Immunotherapy, ClinicalAllergy and Immunology Series 21, Lockey and Ledfors, 4^(th) ed., page162-164).

All the products on the market today for immunotherapy of patients withhouse dust mite allergy comprises aqueous allergen extracts containinggroup 1 and group 2 mite allergen. Many products contain extracts ofboth Dermatophagoides pteronyssinus and Dermatophagoides farinae.

Mite allergen extracts are usually prepared by cultivation of mites in asuitable mite medium followed by extraction of the whole mite culture orextraction of a mite body fraction thereof, Allergens and AllergyImmunotherapy, Fourth Edition, Clinical Allergy and Immunotherapy,series 21, Ed: R. F. Lockey and D. K. Ledfors, page 286.

In the manufacture of allergen extract it is important to control thevariability and to achieve consistency and reproducibility for optimalsafety and efficacy in a subsequent clinical setting. Standardization ofallergen extract are is a complex matter and in principle involves theentire production chain of processes including establishment of robustand reproducible manufacturing procedures, see Allergy Methods andProtocols, Jones and Lympany (2008), chapter 12 and 13. Because of theIgE binding capacity of an allergen extract is related to the content ofone or a few major allergens the general standardization strategynormally involves standardizing the extract in respect to content of aselection of these major allergens further to overall IgE bindingpotency and composition. Relevant method for assays to access allergenextracts are well known in the art and include immunoelectrophoresisassay such as CIE, CRIE, FRIE, CLIE, quantification methods using mono-or polyspecific antibodies such as SRID, RIS, QIE as well as methods ofmaking standardization references. International standards exist to alimited extent, one example being the FDA; CBER reference provided forextract manufactures to standardize against and label their product withresulting in a biopotency label of AI or BAU (Allergy Units orBioequivalent Allergy Units, see FDA DOCKET NO. 94N-00121 (1993)“Methods of the allergenics products testing Laboratory).

There is considerable batch to batch variation in the amount by weightof group 1 and group 2 allergens in allergen extracts and the amount byweight of group 1 allergen in these allergen extracts is typicallyhigher than the amount of group 2 allergen by weight.

It is well known that the house dust mite group 1 and group 2 allergensare found in both the bodies of the mite and in the faecal particlesproduced by the mites during cultivation. It is also known that group 1allergen is associated with the faecal particles and the group 2allergen is associated with the mite bodies; see Batard el al. Int ArchAllergy Immunol 2006, 140, p. 295-305.

As mentioned above there is a considerable batch to batch variation inthe content of mite group 1 and mite group 2 allergens in batches ofwhole mite culture extracts, including extracts of mite bodies, whichmakes it very difficult to prepare mite extracts with a constant andpredetermined level of group 1 and group 2 allergen. This is of course aserious problem in the industrial or large scale production of apharmaceutical product which should contain as closely as possible thesame amount of active ingredient(s) in every batch.

Therefore improved processes and product(s) are important to maximizeproduction efficiency as well as provide high quality products forend-user efficacy and safety.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a pharmaceutical productcomprising an allergen extract or an allergoid thereof, which comprisesat least one extract of mite bodies selected from the following groupsa)-b):

-   -   a) An extract of Der p mite bodies,    -   b) An extract of Der f mite bodies,    -   and at least one extract of mite cultures selected from the        following groups c)-g):    -   c) An extract of Der p faecal particles,    -   d) An extract of Der f faecal particles,    -   e) An extract of Der f whole mite culture,    -   f) An extract of an Der p whole mite culture,    -   g) a combination of extracts c) to f).

The present invention also relates to a pharmaceutical productcomprising an allergen extract or an allergoid thereof, which comprisesat least one extract of mite bodies selected from the following groupsa)-b):

a) An extract of Der p mite bodies,

b) An extract of Der f mite bodies,

and at least one extract of mite faecal particles selected from thefollowing groups c)-d):

c) An extract of Der p faecal particles,

d) An extract of Der f faecal particles.

The invention also relates to a mite body fraction comprising more than70% v/v mite bodies, more preferred more than 75% v/v mite bodies, morepreferred more than 80% v/v mite bodies, preferably more than 85% v/vmite bodies, preferably more than 90% v/v mite bodies, preferred morethan 95% v/v mite bodies and most preferred more than 98% v/v mitebodies.

The invention also relates to a mite faecal particle fraction comprisingmore than 70% v/v faecal particles, preferably more than 75% v/v faecalparticles, preferably more than 80% v/v faecal particles, preferablymore than 85% v/v faecal particles, preferably more than 90% v/v faecalparticles, more preferred more than 95% v/v faecal particles, mostpreferred more than 98% v/v faecal particles.

The invention also relates to a method for the manufacture of a miteallergen extract comprising Der p 1 and Der p 2 allergen, which methodcomprises the following steps:

a) Isolating fraction(s) of Der p mite bodies from cultures of Der pmites, where said fraction(s) comprises more than 70% v/v Der p mitebodies, more preferred more than 75% v/v Der p mite bodies, morepreferred more than 80% v/v Der p mite bodies, preferably more than 85%v/v Der p mite bodies, preferably more than 90% v/v Der p mite bodies,preferred more than 95% v/v Der p mite bodies and most preferred morethan 98% v/v Der p mite bodies;

b) Optionally combining several of said fraction(s) of Der p mitebodies; and

c) Extracting allergens from said Der p mite body fraction(s) obtainedin a step a) or a step b) as above to obtain said mite allergen extract.

The invention also relates to a method for the manufacture of a miteallergen extract comprising Der f 1 and Der f 2 allergens, which methodcomprises the following steps:

a) Isolating fraction(s) of Der f mite bodies from cultures of Der fmites, where said fraction(s) comprises more than 70% v/v Der f mitebodies, more preferred more than 75% v/v Der f mite bodies, morepreferred more than 80% v/v Der f mite bodies, preferably more than 85%v/v Der f mite bodies, preferably more than 90% v/v Der f mite bodies,preferred more than 95% v/v Der f mite bodies and most preferred morethan 98% v/v Der f mite bodies;

b) Optionally combining several of said fraction(s) of Der f mitebodies; and

c) Extracting allergens from said Der f mite body fraction(s) obtainedin a step a) or a step b) as above to obtain said mite allergen extract.

The invention also relates to a method for the manufacture of apharmaceutical composition for the treatment and/or prevention ofallergy or allergic asthma caused by house dust mites which comprise anallergen extract or allergoids thereof having a predetermined andcontrolled content of allergens selected from Der f 1, Der f 2, Der p 1and Der p 2 allergens and comprising the following steps:

a) Fractions of Der p mite bodies and/or fractions of Der p mite faecalparticles are isolated from Der p cultures of mites, and/or fractions ofDer f mite bodies and/or fractions of Der f mite faecal particles areisolated from Der f mite cultures; optionally

b) Combining several fractions of Der p mite bodies, optionallycombining several fractions of Der p faecal particles, optionallycombining several fractions of Der f mite bodies and optionallycombining several fractions of Der f faecal particles;

c) Extracting allergens from mite body faction(s) obtained in a step a)or a step b) as above and/or extraction of allergens from mite faecalparticle fraction(s) obtained in a step a) or a step b) as above; andthereafter

d) Measuring the concentration (w/v) of group 1 and group 2 allergen inmite allergen extracts obtained in a step c) as above; and optionally

e) mixing one or more extract(s) of mite bodies with one or moreextract(s) of faecal particles as obtained in a step c) as above toobtain a mixture of allergen extracts with a predetermined concentration(w/v) of group 1 to group 2 allergen, and optionally

f) converting the extract to allergoids of extract.

The invention also relates to an allergen extract of a series ofallergen extracts for the use in a pharmaceutical allergen product forthe treatment and/or prevention of allergy and allergic asthma caused byhouse dust mites, each allergen extract comprising at least one extractof mite bodies, and at least one extract of mite faecal particles, or amite body extract from a fraction of mite bodies having less than 70%v/v mite bodies or an whole mite culture extract, wherein said allergenextract has a predetermined ratio of group 1 and group 2 allergen, andwherein the variance coefficient of said ratio is no more than 25, 20,15, 10, or 7.5% for the series of extracts.

The invention also relates to an allergen extract of an series ofallergen extracts for the use in a pharmaceutical allergen product forthe diagnosis of allergy caused by house dust mites, each allergenextract comprising at least one extract of mite bodies and at least oneextract of mite faecal particles, or a mite body extract from a fractionof mite bodies having less than 70% v/v mite bodies or an whole miteculture extract, wherein said allergen extract has a predetermined ratioof group 1 and group 2 allergen, and wherein the variance coefficient ofsaid ratio is no more than 25, 20, 15, 10, or 7.5% for the series ofextracts.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a set-up as further described in Example 2.

DEFINITIONS

As used herein “mite” refers to house dust mites. Examples of such mitesare Dermatophagoides farinae and/or Dermatophagoides pteronyssinusand/or Euroglyphus maynei or other important species belonging to thefamily of Pyroglyphidae.

As used herein “Der p” means “Dermatophagoides pteronyssinus”, “Der p 1”means “group 1 allergen of Der p” and “Der p 2” means “group 2 allergenof Der p”.

As used herein “Der f” means “Dermatophagoides farinae”, “Der f 1” means“group 1 allergen of Der f ” and “Der f 2” means “group 2 allergen ofDer f”.

The group 1 allergens and the group 2 allergens of the two mite speciesDer f and Der p exist naturally in a number of isoforms, see e.g.www.allergen.org under allergen sources (AnimaliaAnthropoda/Astigmata/Dermatophagoides farinae or Dermatophagoidesfarinae). As used herein “group 1 allergen”, “group 1”, “group 2allergen” and “group 2” includes all naturally occurring isoforms ofeach of the group 1 and group 2 allergens including those mentioned onwww.allergen.org.

As used herein “culture of mites” or “mite culture”, “whole miteculture” and “whole culture”, “Der p mite culture” and “Der f miteculture” or “cultures of Der p mites” and “cultures of Der f mites”means the material obtained after growth of Der p or Der f on a suitablemedium and contains the whole culture including mites, parts of mites,faecal particles, eggs, mite media components etc.

As use herein “mite culture media or medium” or “mite media or medium”means a diet suitable for growing house dust mites.

As used herein “extract of mite bodies”, “extract of Der p mite bodies”and “extract of Der f mite bodies” means “extract of one fraction ormore combined fractions of mite bodies”, “extract of one fraction ormore combined fractions of Der p mite bodies” and “extract of onefraction or more combined fractions of Der f mite bodies” respectively.

As used herein “extract of mite faecal particles”, “extract of faecalparticles”, “an extract of Der p faecal particles” and “extract of Der ffaecal particles” means “extract of one or more combined fraction(s) ofmite faecal particles”, “extract of one fraction or more combinedfraction(s) of faecal particles”, “extract of one or more combinedfraction(s) of Der p faecal particles” and “extract of one or morecombined fraction(s) of Der f faecal particles”, respectively.

As used herein “allergen extract” and “allergen extract (I)” are usedinterchangeably and mean an extract of at least one “mite body extract”or “faecal particle extract” optionally in combined with further miteextracts and in any combination. As used herein “ fraction(s) of mitebodies”, means one or more combined fraction(s) of mite cultures thatcontain more than 50% v/v mite bodies, “fraction(s) of Der p mitebodies” means one or more combined fraction(s) of mite Der p culturesthat contain more than 50% v/v Der p mite bodies, and “fraction(s) ofDer f bodies” means one or more combined fraction(s) of Der f mitecultures that contain more than 50% v/v Der f bodies.

As used herein “fraction(s) of mite faecal particles” and “fraction(s)of faecal particles”, means one or more combined fraction(s) of mitecultures that contain more than 50% v/v mite faecal particles,“fraction(s) of Der p faecal particles” means one or more combinedfraction(s) of Der p mite cultures that contain more than 50% v/v Der pfaecal particles, and “fraction(s) of Der f faecal particles” means oneor more combined fraction(s) of Der f mite cultures that contain morethan 50% v/v Der f faecal particles.

As used herein “(s)” as the last part of a word means the word in eithersingular or plural form.

As used herein “treatment” means “curing or alleviation of allergicsymptoms”.

As used herein “fast dissolving sublingual tablet” refers to compressedand non-compressed dosage forms which disintegrate in less than about 90seconds, preferably in less than about 60 seconds, preferably in lessthan about 30 seconds, more preferably in less than about 20, even morepreferably in less than about 10 seconds in the oral cavity, even morepreferred in less than about 5 seconds, and most preferably in less thanabout 2 seconds after being received under the tongue in the oralcavity. Such fast dissolving sublingual tablets are described in e.g. WO04/77994.

As used herein “CV” shall mean the coefficient of variation expressed inpercentage.

DETAILED DESCRIPTION OF THE INVENTION

The pharmaceutical products of the invention are suitable for thetreatment and/or prevention of house dust mite allergy and allergicasthma caused by house dust mites.

As mentioned above there is a considerable batch to batch variation inthe content of mite group 1 and mite group 2 allergens in batches ofwhole mite culture extracts, including extracts of mite bodies, whichmakes it very difficult to prepare mite extracts with a constant andpredetermined level of group 1 and group 2 allergen.

It has now been found that by preparing several, such as two extracts,for each batch of mite culture, for example by isolating a fraction ofthe mite culture containing primarily mite bodies and a fraction of themite culture comprising mainly mite faecal particles followed byseparate extraction of the mite body fraction and the mite faecalparticles fraction provides the ability to control of the ratio of group1 to group 2 allergen in the final allergen extract by mixing the mitebody extract with mite faecal particles extract until the desired weightratio is reached.

It has also been found that if the mite culture obtained by cultivationof mites is purified to produce a mite body fraction containing morethan 70% v/v or more preferred more than 80% v/v, more preferred morethan 85% v/v, more preferred more than 90% v/v, more preferred more than95% v/v or more preferred more than 98% v/v mite bodies, extraction ofthis purified mite body fraction will normally produce a mite bodyextract which contain equal amount by weight of group 1 and group 2allergen or more group 2 allergen than group 1 allergen.

A mite body extract with more group 2 than group 1 allergen by weightmay be supplemented with extract of mite faecal particles which normallycontain more group 1 allergen than group 2 allergen by weight to createa mixture of extracts with predetermined and if desired more equalamounts by weight of group 1 and group 2 allergen.

Examples of extracts, which normally contain more group 1 allergen thangroup 2 allergen by weight and which may be used to supplement with, isan extract of a whole mite culture or a body fraction(s) having lessthan 70% v/v of mite bodies.

A mite body extract with more group 2 than group 1 allergen by weightmay also be supplemented with extract of whole mite culture or a mitebody fraction having less than 70 v/v % mite bodies, both which normallycontain more group 1 allergen than group 2 allergen by weight to createa mixture of extracts with predetermined and if desired more equalamounts by weight of group 1 and group 2 allergen.

It is possible by mixing extracts of mite bodies and other fractions,preferably faecal particles fractions to obtain a mixture of extractswhich contain a broad range of group 1 to group 2 weight ratios, such asfrom 1:0.6 to 1:1.6, 1:0.7 to 1:1.5, 1:0.8 to 1:1.4, 1:0.9 to 1:1.3,1:0.95 to 1:1.2, 1:0.98 to 1:1.15, including a mixture which containequal amounts by weight of group 1 and group 2 allergens, i.e. a 1:1:1:1mixture of Der p 1, Der p 2, Der f 1 and Der f 2.

In one aspect, the invention relates to a method for the manufacture ofa mixture of mite allergen extracts for a pharmaceutical product havinga predetermined, controlled amount by weight of Der f 1, Der f 2, Der p1 and Der p 2 allergens.

The invention also relates to a pharmaceutical product comprisingallergen extracts selected from at least one extract of mite bodies andoptionally at least one extract of mite faecal particles selected from:a) an extract of Der p mite bodies, b) an extract of Der p faecalparticles, c) an extract of Der f mite bodies, and d) an extract of Derf faecal particles where the major allergens Der p 1, Der p 2, Der f 1and/or Der f 2 is present in predetermined, controlled amounts.

In some embodiments the extracts of the invention are characterized bycontaining more group 2 allergen by weight compared to products on themarket. The inventors of the present application have provided anindustrial or large scale process for producing mite allergen extractswith this altered weight ratio between group 1 and group 2 allergen.

It has been found that if separate extracts of both mite bodies and mitefaecal particles are prepared for each of the two mite species, and asufficiently pure mite body fraction (and mite faecal particlesfraction) is used for extraction it is possible to create mite extractswith a weight ratio of group 1 to group 2 allergen that is between 1:0.6to 1:1.6 for each mites species.

By combining the extracts of both species a weight ratio between Der p1, Der p 2, Der f 1 and Der f 2 that is 1:1:1:1 may be achieved.

These products are advantageous because the content of allergens in thepharmaceutical products is well controlled.

Previously mite allergen extracts typically contained more group 1allergen than group 2 allergen by weight which may have caused a poortreatment of allergic patients that are particularly sensitized to group2 allergen.

According to the invention, an industrial process for the preparation ofextracts with a higher amount by weight of group 2 allergen than group 1allergen than normally found in extracts have been provided.

Suitably, the pharmaceutical product as above comprises at least oneextract of mite bodies and at least one extract of mite faecal particlesselected from the groups a)-d) above.

Preferably the mite allergen extract comprises an extract of Der p mitebodies, an extract of Der p faecal particles, an extract of Der f mitebodies and an extract of Der f faecal particles.

According to further embodiments of the invention the mite allergenextract comprise:

-   -   An extract of Der p mite bodies and an extract of Der p faecal        particles and optionally no extracts of Der f mite bodies and no        extracts of Der f faecal particles; or    -   An extract of Der f mite bodies and an extract of Der f faecal        particles and optionally no extracts of Der p mite bodies and no        extracts of Der p faecal particles; or    -   An extract of Der p mite bodies and an extract of Der f mite        bodies and optionally no extract of mite faecal particles; or    -   An extract of Der p mite bodies and no extract of Der f mite        bodies and optionally no extract of mite faecal particles; or    -   An extract of Der f mite bodies and no an extract of Der p mite        bodies extract and optionally no extract of mite faecal        particles.

In a preferred embodiment of the invention the extract of Der p mitebodies is prepared from one or more fraction(s) of Der p mite bodieswhere extraction of the fraction(s) of Der p mite bodies results in anextract comprises equal amounts by weight of group 1 and group 2allergen or more group 2 allergen than group 1 allergen by weight and/orthe extract of Der f mite bodies is prepared from one or morefraction(s) of Der f mite bodies and where extraction of the fraction(s)of Der f mite bodies results in an extract comprises equal amounts byweight of group 1 and group 2 allergen or more group 2 allergen thangroup 1 allergen by weight.

In a preferred embodiment

-   -   the extract of Der p mite bodies is suitably prepared from one        fraction or combined fractions of Der p mite cultures said        fraction(s) comprising more than 70% v/v Der p mite bodies, more        preferred more than 75% v/v Der p mite bodies, more preferred        more than 80% v/v Der p mite bodies, preferably more than 85%        v/v Der p mite bodies, preferably more than 90% v/v Der p mite        bodies, preferred more than 95% v/v Der p mite bodies and most        preferred more than 98% v/v Der p mite bodies, and/or    -   the extract of Der f mite bodies is suitably prepared from one        fraction or combined fractions of mite Der f cultures said        fractions comprising more than 70% v/v Der f mite bodies,        preferably more than 75% v/v Der f mite bodies, preferably more        than 80% v/v Der f mite bodies, preferably more than 85% v/v Der        f mite bodies, preferably more than 90% v/v Der f mite bodies,        more preferred more than 95% v/v Der f mite bodies, and        preferably more than 98 ° A) v/v Der f mite bodies, and/or    -   the extract of Der p faecal particles is suitably prepared from        one fraction or combined fraction(s) of Der p mite cultures said        fraction(s) comprising more than 70% v/v Der p faecal particles,        preferably more than 75% v/v Der p faecal particles, preferably        more than 80% v/v Der p faecal particles, preferably more than        85% v/v Der p faecal particles, preferably more than 90% v/v Der        p faecal particles, more preferred more than 95% v/v Der p        faecal particles, most preferred more than 98% v/v Der p faecal        particles and/or    -   the extract of Der f faecal particles is suitably prepared from        one fraction or combined fraction(s) of Der f mite cultures said        fraction(s) comprising more than 70% v/v Der f faecal particles,        preferred more than 75% v/v Der f faecal particles, preferred        more than 80% v/v Der f faecal particles, preferably more than        85% v/v Der f faecal particles, preferably more than 90% v/v Der        f faecal particles, preferred more than 95% v/v, and more        preferred more than 98% v/v Der f faecal particles.

In one embodiment of the invention, the faecal fraction is not so pureas the body fraction.

In a preferred embodiment each of the fractions or combined fractionsabove comprises below 10% v/v mite culture media particles.

The mite body fraction or combined mite body fractions may contain up to20% v/v mite parts (legs, eggs etc), medium and faecal particles (i.e.anything that is not mite bodies or contaminants), provided the amountof medium particles is below 10% v/v.

The mite faecal particles fraction or combined mite faecal particlesfractions may contain up to 20% v/v mite parts (legs, eggs etc) andmedia particles (i.e. anything that is not faecal particles orcontaminants), provided the amount media particles is below 10% v/v.

In a further embodiment each of the fractions or combined fractions iscomprised of no more than a total of 1.0% w/w of detectable foreignmaterials. Mite faecal particles, mite parts, mite bodies or mite mediaare not considered to be foreign materials.

The volumetric percentages of mite bodies and mite faecal particles inthe mite body and faecal fractions of particles are determined bymicroscopy. The number of particles (e.g. mite body or faecal particle)is counted and expressed as % v/v by multiplication with a figure whichis the average volume for the particle in question (e.g. mite body orfaecal particle).

Microscopic Count Performed on Mite Body Fractions:

A sample is weighed out and suspended in a glycerol containing solution(25% glycerol, 5% Igepal). The sample is poured onto a gridded filterwhile under vacuum in order to remove the liquid and immobilise theparticles on the filter. The filter is then dried to increase visibilitywhen using the microscope. Grids to be counted are chosen at random andall particles within a chosen grid are counted. Grids are chosen untilat least 800 particles have been counted. The above procedure may beperformed on several such as two, three, four or five independentsamples. When calculating the results, the variation between the sampleshas to meet a desired acceptance criteria (such as for example more than80 vol/vol % whole mite bodies, and/or less than 10 vol/vol % mediaparticles) for the assay in order to ensure consistency between thesamples. If the results from the samples are consistent, then the countsare averaged giving the assay result.

The non-volumetric counts are translated into volumes by usingpredetermined assigned volumes for mite bodies, faecals, and mediumparticles.

Microscopic Count Performed on Faecal Fractions:

A sample is weighed out, placed in a gridded counting chamber and mixedwith paraffin oil to give an even layer. Grids to be counted are chosenat random and all particles within a chosen grid are counted. Grids arechosen until at least 800 particles have been counted. The aboveprocedure may be performed on several such as two, three, four or fiveindependent samples. When calculating the results, the variation betweenthe samples has to meet an acceptance criteria (such as for example morethan 80 vol/vol % whole mite bodies, and/or less than 10 vol/vol % mediaparticles) for the assay in order to ensure consistency between thesamples. If the results from the samples are consistent, then the countsare averaged giving the assay result The non-volumetric counts aretranslated into volumes by using predetermined assigned volumes for mitebodies, faecals, and medium particles.

The volumes assigned to mite bodies, faecal particles, mite mediumparticles are as follows:

Der f mite bodies: 21132000 cubed micrometers

Der p mite bodies: 12926000 cubed micrometers

Mite medium particles in body fractions: 6721000 cubic micrometers

Mite medium particles in faecal fractions: 10000 cubic micrometers

Faecal particles: 19000 cubic micrometers

As mentioned above the present invention is based on the finding that bypurifying the mite body fraction(s) so that they contain more than 70%v/v of mite bodies, more preferred more than 80% v/v, more preferredmore than 85% v/v, more preferred more than 90% v/v, more preferred morethan 95% v/v or more preferred more than 98% v/v it is possible toprepare a mite body extract from such mite body fraction or combinedfractions where the amount by weight of group 1 allergen in the extractis lower than the amount of group 2 allergen by weight or theconcentration of group 1 and group 2 allergen by weight is equal.

According to a preferred embodiment, the amount by weight of group 1allergen is lower than the amount by weight of group 2 allergen in themite body extract(s) and the amount by weight of group 1 allergen ismuch higher than the weight group 2 allergen in the extracts of mitefaecal particles.

Suitably the weight ratio of group 1 to group 2 allergen in the allergenextract is closer to the weight ratio of group 1 allergen to group 2allergen in the mite body extracts than the weight ratio of group 1 togroup 2 allergen in the mite faecal particles extracts which is used forthe preparation thereof.

According to the invention it is possible to alter the weight ratio ofgroup 1 to group 2 allergen to more equal amounts by weight in theextract, than in the extract of mite bodies and mite faecal particlesfrom which they were prepared. This was not possible previously wheretypical batches of both mite bodies and the faecal particles containmore group 1 allergen than group 2 allergen by weight.

A pharmaceutical product of the invention is provided, comprisingallergen extract, which comprises allergens selected from Der f 1, Der f2, Der p 1 and Der p 2 and where the allergen present in the lowestconcentration (w/v) in the allergen extract, is present in aconcentration which is above 50% of the concentration of the allergenselected from Der f 1, Der f 2, Der p 1 and Der p 2 present in thehighest concentration (w/v), more preferred above 60% of theconcentration of the allergen selected from Der f 1, Der f 2, Der p 1and Der p 2 present in the highest concentration (w/v), more preferredabove 70% of the concentration of the allergen selected from Der f 1,Der f 2, Der p 1 and Der p 2 present in the highest concentration (w/v),more preferred above 80% of the concentration of the allergen selectedfrom Der f 1, Der f 2, Der p 1 and Der p 2 present in the highestconcentration (w/v), more preferred above 85% of the concentration ofthe allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2 presentin the highest concentration (w/v), more preferred above 90% of theconcentration of the allergen selected from Der f 1, Der f 2, Der p 1and Der p 2 present in the highest concentration (w/v), more preferredabove 95% of the concentration of the allergen selected from Der f 1,Der f 2, Der p 1 and Der p 2 present in the highest concentration (w/v),preferred above 98 ° A) of the concentration of the allergen selectedfrom Der f 1, Der f 2, Der p 1 and Der p 2 present in the highestconcentration (w/v), or most preferred above 99% of the concentration ofthe allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2 presentin the highest concentration (w/v).

One daily dose of a product of the invention suitably contain from 1-25μg or more, 1-15 μg or more, 1-12 μg or more, 1-10 μg or more, 2 to 10μg or more preferred from 3 to 8 μg Der p 1, from 3 to 8 μg Der p 2,from 3 to 8 μg Der f 1 and from 3 to 8 μg Der f 2.

In a preferred embodiment the daily dose of group 1 and group 2allergens in a sublingual product of the invention is suitably between 3and 4 μg, more preferred around 3.6 μg of Der p 1, Der p 2, Der f 1and/or Der f 2.

In another embodiment the daily dose of group 1 and group 2 allergens ina sublingual product of the invention suitably between 6 and 8 μg, morepreferred around 7.2 μg of Der p 1, Der p 2, Der f 1 and/or Der f 2.

The pharmaceutical product as above may be formulated as a compressed ornon-compressed sublingual tablet (e.g. as in WO 04/77994), a liquidsublingual product (e.g. as in WO 07/051476) or a liquid product forinjection optionally adjuvanted with aluminium hydroxide (e.g. as in WO10/043675). Preferably the pharmaceutical product is a fast dissolving,sublingual tablet. Suitably the tablets are in the form of tablets,capsules, lozenges or caplets.

The allergen extract may be present in the form of allergoids in thepharmaceutical products described above.

The present invention also relates to a method for the manufacture of amite allergen extract for a pharmaceutical product said extract having apredetermined, controlled and if desired a more equal weight ratio ofDer f 1, Der f 2, Der p 1 and/or Der p 2 allergen.

The invention also relates to a method for the manufacture of a miteallergen extract or allergoids thereof for a pharmaceutical product asdescribed above wherein said allergen extract having a predetermined andcontrolled amount by weight of allergens selected from Der f 1, Der f 2,Der p 1 and Der p 2 allergens and comprising the following steps a), andc)-d) and optionally b), e) and/or f):

a) Fractions of Der p mite bodies and/or fractions of Der p mite faecalparticles are isolated from Der p cultures of mites, and/or fractions ofDer f mite bodies and/or fractions of Der f mite faecal particles areisolated from Der f mite cultures;

b) Optionally combining several fractions of Der p mite bodies,optionally combining several fractions of Der p mite faecal particles,optionally combining several fractions of Der f mite bodies andoptionally combining several fractions of Der f mite faecal particles;

c) Extracting allergens from mite body fraction(s) obtained in a step a)or a step b) as above and/or extraction of allergens from faecalparticle fraction(s) obtained in a step a) or a step b) as above; andthereafter

d) Measuring the concentration (w/v) of group 1 and group 2 allergen inmite allergen extracts obtained in a step c) as above; and optionally

e) Mixing one or more extract(s) of mite bodies with one or moreextract(s) of mite faecal particles as obtained in a step c) as above toobtain a mixture of allergen extracts with a predetermined amount andweight ratio of group 1 to group 2 allergen, and optionally

f) Converting the allergen extract to an allergoid thereof.

According to the invention extracts of fractions of mite bodies andextracts of fractions of mite faecal particles may be combined or mixed,however fraction(s) of mite bodies are never combined with faecalparticles fraction(s).

Suitably one or more extract(s) of mite bodies and one or moreextract(s) of faecal particles are mixed to obtain a mixture of allergenextracts.

Preferably the mixture of mite allergen extract comprises extract(s) ofDer p mite bodies, extract(s) of Der p faecal particles, extract(s) ofDer f mite bodies and extract(s) of Der f 2 faecal particles.

To prepare the extract more than one fraction of mite bodies may becombined according to step b) followed by extraction according to stepc).

Also more than one fraction of mite faecal particles may be combinedaccording to step b) followed by extraction according to step c).

Alternatively, a fraction of mite bodies obtained in step a) issubjected to extraction and/or a fraction of mite faecal particlesobtained in step a) is subjected to extraction.

Suitably one or more extract(s) of mite bodies obtained in a step c) ismixed in a step e) with one or more extract(s) of mite faecal particlesobtained in a step c).

Suitably one extract of mite bodies obtained in a step c) is mixed in astep e) with one extract of mite faecal particles obtained in a step c).

In the method as above one or more extract(s) of mite bodies obtained ina step c) may be mixed in step e) with one extract of mite faecalparticles obtained in a step c).

Alternatively, one extract of mite bodies obtained in a step c) may bemixed in a step e) with one or more extract(s) of mite faecal particlesobtained in a step c).

As used herein, “mixing of extracts” include mixing of both wholebatches of extracts as well as parts of whole batches.

According to a preferred embodiment of the invention the mite bodyextract(s) are supplemented with an amount of faecal particles extractwhich is sufficient to achieve the desired weight ratio of group 1allergen to group 2 allergen in the mixture of mite allergen extracts.

In another preferred embodiment of the invention, the mite body extractalready has an almost equal amount of group 1 and group 2 allergen byweight and need not be mixed with mite faecal particles extract.

The coefficient of variation (CV) is calculated as the ratio of thestandard deviation σ to the mean μ:

$c_{v} = \frac{\sigma}{\mu }$

It is sometimes expressed as percentage, in which case the CV ismultiplied by 100.

The CV is used to describe the improved batch-to batch consistency ofthe extract and pharmaceutical products (therapeutic or diagnostic)according to the invention.

Confidence Intervals (CI) such 95% may be used.

It is sometimes expressed as percentage, in which case the CV ismultiplied by 100.

The CV is used to describe the improved batch-to batch consistency ofthe extract and pharmaceutical products (therapeutic or diagnostic)according to the invention.

Confidence Intervals (CI) such 95% may be used.

Cultivation of Mites:

A number of diets suitable for growth of the mites are known and therehave been numerous studies of the effectiveness of different culturemedium ingredients such as yeast, albumins, feed for animals, feed forfish (Tetramin), derivatives of shed human skin scales, pork skins,brine shrimp eggs, wheat germs, rat food, dog food, powdered animalliver, hydrolysate of milk proteins, vitamins, minerals, proteinhydrolysate and soya flour.

Mite diets are for example described in International Journal ofAcarology, Vol. 8, Issue 3 (1982), p. 189-192, Rodrigues, RecentAdvances in Acarology, Vol. 11 (1979), 211-216, Batard et al., Int ArchAllergy Immunol. 140 (2006) p. 295-305, Yi et al.; Asian Pacific Journalof Allergy and Immunology, 17 (1999), p. 189-194, Arlian et al., J.Allergy. Clin. Immunol, No. 3 (1987), p. 457-66, and EP patentNo.1236394.

One example of a medium for rearing mites comprising a 1:1 mix of yeastand dried Daphnia is described in Dust Mites by Matthew Colloff (2009),p. 270. Another example of a medium for rearing mites is as described inEP patent No. 1236394.

It is preferred to use a protein hydrolysate as well as otheringredients that are of low allergenic risk in order to reduce thepossibility of anaphylactic reactions in allergen sensitized patients.It is further preferred to use mite medium that do not contain animal orhuman protein.

Suitably the mite medium is used in the form of particles which are ofsufficient size and integrity to withstand the rigors of downstreamprocessing and be easily eliminated through the primary screeningprocess.

The two mite species Der p and Der f are always grown in separatecontainers but may be grown on the same mite medium. Examples ofcontainers include glass or plastic containers of suitable size andshape such as fly-bottles. Disposable containers for culturing mites arefor example described in WO2008/119762.

The mites are cultivated in containers with culture medium and areplaced in controlled environment controlling the temperature andhumidity of the environment surrounding the growing mite cultures.Suitable conditions for Der p and Der f are for example described in anumber of articles such as by Fain et al (ISBN 90-71868-12-5) and byCrowther: Exp Appl Acarol. 2007; 41(1-2):61-86.

One suitable set of parameters for the cultivation conditions for eachof the mite species is provided in table 1a below.

TABLE 1a Der f Der p Relative 65% ± 5% RH 70% ± 5% RH humidityTemperature 25° C. ± 5° C. such as 25° C. ± 7° C. such as 30° C. ± 27.5°C. ± 2° C. 2° C. Cultivation 42-70 such as 42-56 42-70 such as 42-56days time days

Microscopic counts for growth and viability are performed during thecultivation or order to identify the optimal time for harvesting themites.

Killing of the culture may be performed by freezing. Mites can also bekilled by e.g. suffocation with e.g. acetone. Storage containers withkilled mite culture are stored at −20° C. until further processing.

Purification of Mite Culture:

The mite cultures are thawed and dried and sieved to obtain fractions ofthe mite culture containing components of different sizes. Drying may becarried out by airflow until moisture is less than 20%, more preferred15%

The mite culture may be further purified and mite body or faecalfractions isolated using separation techniques including sieving (e.g.using meshes of 1 mm, 500 μm, 250 μm, 100 μm on a sieve-shaker),saturated sodium chloride suspensions, and ethanol suspension, see e.g.Dust Mites by Matthew Colloff (2009), p. 271.

In one embodiment the fraction(s) of mite bodies and the fraction(s) ofmite faecal particles may be prepared by sieving of the mite culture toproduce four fractions:

a) Large medium particles >350 μm

b) Mite body fraction 350-90 μm

c) Faecal agglomerates and mite parts 90-50 μm

d) Faecal particles <50 μm

It is within these size ranges the different components of the miteculture, such as the mite bodies, the faecal particles and mediumparticles etc. are usually found. Mite culture fractions isolated withina size range that differ from the size ranges above may also be usefulaccording to the invention as long as it is possible to prepare mitebody fractions and faecal particles that are sufficiently pure and insufficient amount.

Fraction a) and c) are discarded. Fraction d) may be used directly inthe extraction process and fraction b) is subjected to furtherpurification before extraction.

Purification of Mite Body Fraction:

A method for purification of mite bodies from medium particles byflotation in a suitable liquid have been described previously, GB patentno. 1318560 on page 3. J. Med. Entomol. (1979), Vol. 16, No. 2, p.128-132 describes flotation in alcohol (ethanol) for the separation ofmite bodies from mite media particles.

It has now been found that density centrifugation, optionally repeated,of the mite body fraction in a suitable liquid produces a mite bodyfraction of high purity which when extracted produces an extract withmore group 2 allergen than group 1 allergen weight, or an equal amountby weight of group 1 and group 2 allergen.

Thus the present invention also relates to a method for the isolationand purification of mite bodies comprising the steps:

i) Isolating mite body fraction(s) from Der p and/or Der f miteculture(s) and

ii) Subjecting one or more Der p mite body fraction(s), or one or moreDer f mite body fraction(s), to density centrifugation in a first liquidmedium having a higher density than the mite bodies and a lower densitythan the culture media at the temperature used during thecentrifugation; and

iii) Collecting the mite bodies from the surface of said first liquidmedium and optionally repeating step ii) and iii) until the desiredpurity of the mite bodies have been achieved, optionally followed by

iv) Washing of the mite bodies to remove said first liquid with a secondliquid that does not extract group 1 and group 2 allergen from the mitebodies at the temperature used during the washing step.

The present invention also relates to a method for the isolation andpurification of mite bodies comprising the steps:

i) Isolating mite body fraction(s) from Der p and/or Der f miteculture(s) and

ii) Subjecting one or more Der p mite body fraction(s), or one or moreDer f mite body fraction(s), to density centrifugation in a first liquidmedium having a higher density than the mite bodies and a lower densitythan the culture media at the temperature used during thecentrifugation; and

iii) Collecting the mite bodies from the surface of said first liquidmedium optionally followed by

iv) Washing of the mite bodies to remove said first liquid with a secondliquid that does not extract group 1 and group 2 allergen from the mitebodies at the temperature used during the washing step.

Centrifugation parameter settings e.g. Relative Centrifugation Force(RCF), velocity (RPM; Round Per Minutes), centrifugation time, rates ofliquid to mite body fraction(s), temperature are well known to theskilled person in the art and may have to be adjusted depending oncentrifugation equipment and degree of automation. Useful RCF are inrange of 500-5000 RCF. Setting include 1000-7000 RPM and centrifugationtime of 1-20 minutes or more preferred 1500-6000 RPM and 2-15 minutes,even more preferred 2000-4000 and 3-12 minutes. Centrifugation cups maybe filled or filled less. The rate of dried mite fraction to liquid(w/w) varies depending on liquid used. Preferably the amount of liquid(weight) used for centrifugation exceeds the amount (weight) of the mitebody fraction by more than a factor 1, more preferably by a factor 3,even more preferred by a factor 5 or above.

Preferably the first and second liquid in the process above does notcontain water (anhydrous) to limit the extraction of allergens from thesource material in the instant source material purification step. Inanother embodiment, the method for the isolation and purification ofmite bodies the step ii) of subjecting one or more Der p mite bodyfraction(s), or one or more Der f mite body fraction(s), to densitycentrifugation in a first liquid medium is performed with a first liquidhaving iia) a higher density than the mite bodies and a lower densitythan the culture media particles and iib) do not extract group 1 andgroup 2 allergen form the mite bodies at the temperature used during thecentrifugation.

Suitable liquids comprise metrizoic acid derivatives (like iohexol,Nycodenz®(N-2,3-dihydroxypropylacetamido-2,4,6-tri-iodo-N-N-bis(2,3-dihydroxypropyl)),polypropyleneglycols (such as ARCOL® (polypropyleneglycol 4000), Silicabased compositions (like RediGrad®).

It is difficult to completely avoid some extraction of group 1 and group2 allergen from the mite bodies during the centrifugation process above.According to the invention, liquids that do not extract group 1 andgroup 2 allergens from the mite bodies are liquids that do not extractgroup 1 allergen and group 2 allergen from the mite bodies at thetemperature used in step ii) and iv) above in an amount that seriouslyaffect the final yield and ratio of group 1 and group 2 allergen.

Extraction of group 1 and group 2 allergen from the mite bodies duringthe centrifugation process may be reduced by carrying out thecentrifugation at a low temperature e.g. freezing temperature.

In a preferred embodiment the liquid having a density higher than themite bodies and lower than the culture media particles is an anhydrousliquid, for example anhydrous glycerol.

The glycerol may be removed from the mite bodies by washing withethanol, preferably anhydrous ethanol.

Suitable step ii) and iii) are repeated once or the centrifugation inglycerol is repeated until the mite body fraction obtained is composedof at least 70% v/v mite bodies, more preferred more than 75% v/v mitebodies, more preferred more than 80% v/v mite bodies, preferably morethan 85% v/v mite bodies, preferably more than 90% v/v mite bodies,preferred more than 95% v/v mite bodies and most preferred more than 98%v/v mite bodies and the mite body fraction contain below 20% v/v miteparts, faecal particles and medium particles and below 10% v/v mediumparticles.

Instead of density centrifugation with glycerol as describe in step ii)and iii) the mite body fraction may be purified by separation withsaturated NaCl as the first liquid until the mite body fraction iscomposed of at least 70% v/v mite bodies, more preferred more than 75%v/v mite bodies, more preferred more than 80% v/v mite bodies,preferably more than 85% v/v mite bodies, preferably more than 90% v/vmite bodies, preferred more than 95% v/v mite bodies and most preferredmore than 98% v/v mite bodies and contain below 20% v/v mite parts,faecal particles and medium particles and below 10% v/v medium particleswithout the need for applying step iv).

Instead of density centrifugation the mite body fraction may be purifiedby sieving until the mite body fraction is composed of at least 70% v/vmite bodies, more preferred more than 75% v/v mite bodies, morepreferred more than 80% v/v mite bodies, preferably more than 85% v/vmite bodies, preferably more than 90% v/v mite bodies, preferred morethan 95% v/v mite bodies and most preferred more than 98% v/v mitebodies and contain below 20% v/v mite parts, faecal particles and mediumparticles and below 10% v/v medium particles.

Mite bodies may in one dimension be more than 400 μm long but maynevertheless be able to pass through a 300 μm and 400 μm sieve when theyare dried before sieving.

Mite bodies have legs and hair and may be separated from mite mediaparticles in sieve shaker with a horizontal circular movement. Themethod takes advantage of the mites ability to form tangles formed withtheir hair and legs when moved horizontally on a sieve. In a sievingtower which is moved horizontally in circles the mite bodies is capturedon top of the 200, 300 and/or the 400 μm mesh sieves and is in this wayseparated from other mite culture particles with a size between 150 and300 μm.

Thus, in an alternative to the centrifugation method the mite body richfraction between 90 and 350 μm isolated in the sieving process above maybe purified by the following method:

1) sieving Der f or Der p mite cultures in a vibrating sieve shaker witha tower of a 300, a 150 and a 80 μm mesh sieve followed by isolation ofa mite body rich fraction between 150 μm and 300 μm and a mite faecalparticles rich fraction below 80 μm, followed by

2) sieving the mite body rich fraction (150 μm and 300 μm) in a sieveshaker with a horizontally and circular movement and with a tower of200, 300 and/or 400 μm mesh sieves and isolating body rich fraction(s)captured by the 200, 300 and/or 400 μm mesh sieves, followed by

3) sieving of the body rich fraction in a sieve shaker having a 3Dthrowing motion and 300, 200, 150 and 100 mesh sieve to produce a mitefraction between 150 and 300 μm mesh and a mite faecal particles extractbelow 100 μm; and

optionally combining the facal particles fraction below 80 μm from thevibrating sieve and the faecal particles fraction below 100 μm from thesieve with the 3D throwing motion and subjecting the combined faecalfraction to sieving in the sieve with the 3D throwing motion and a towerof 100, 80, 50 μm mesh sieves.

Mite body fractions b) obtained above may also be purified by steps 2and 3 in the above process.

Extraction

The fractions of mite bodies and mite faecal particles are extracted inmore or less the same way but in separate processes because the optimalconditions for extraction of mite bodies is not the same as for mitefaecal particles.

In order to extract allergens from the mite bodies and the faecalparticles these, bodies need to be cracked to release the allergens.Cracking can be achieved by applying an external force to a slurry ofmite bodies.

According to the invention it has been found that a high shear mixer isparticularly effective to crack and release the allergens from mitebodies and faecal particles.

Thus, according to one embodiment of the invention the extraction ofallergens is carried out by suspending the mite bodies or the mitefaecal particles in a buffer with a high shear mixer (typically for 15to 30 minutes) and then allowing the extraction to proceed withoutmixing (typically for 1-6 hours).

The release of allergens is different for mite bodies and faecalparticles. The allergens in the faecal particles are extracted after 1hour of extraction whereas at least 3 hours are needed to extractallergens from the mite bodies.

Buffers for extraction of mite allergens are well known in the art.

The allergenic source material (mite body and/or mite faecal particles)may for example be extracted using an extraction buffer comprising 0.15M NaCl, 0.012 M NaHCO₃ and NaOH/HCl/water to adjust pH. The extractionis suitably carried out using a ratio between extraction buffer toallergenic source material of 1:10 (w/w) a pH of 7-8, a temperaturebetween 4-12° C. and an extraction time of between 1-6 hours.

These extracts may be subjected to further purification before they areused in a pharmaceutical product.

The remaining insoluble material may for example be removed from theextract of mite faecal particles by centrifugation and clarification.The mite faecal particles extract may thereafter be subjected toultrafiltration performed in three steps: First concentration by removalof water and other small molecules by tangential flow filtration,followed by diafiltration to remove small molecules and salts andthereafter dry matter adjustment.

The allergen extract may thereafter be clarified by clarificationfiltration.

Further purification of the mite bodies extracts suitably involves thefollowing processes: Separation using diafiltration leaving insolublematter on the membrane and continuously adding water to the extract onthe other side of the membrane. The extract is thereafter subjected toultra filtration and clarification as described above.

The extracts may be stabilized by forming frozen balls of extracts(cryogranules) as described in WO 05/058474.

The concentration of group 1 and group 2 allergen in the extracts ofmite bodies and mite faecal particles may be measured by a number ofmethods conventionally used for the measurement of allergen contentincluding for example ELISA, RID/SRID (Single Radial immunodiffusion) orby MS (mass spectrometry e.g. as described in WO 2007/031080) see alsoabove.

The principle of the RID procedure is that proteins (antigens) appliedin a circular well diffuse outwards forming a concentration gradient inan agarose gel containing the corresponding antibody. The antibody usedin this procedure is mono-specific (i.e. raised against a specificantigen) and polyclonal making it possible to form precipitates ofantigen-antibody complexes. The antibody is present in excess anddiffusion of the antigen will continue until the equivalence point isreached and a ring of antigen-antibody precipitates is formed.Subsequently, the precipitation rings are stained. The area of theprecipitate is measured and the concentration of antigen in unknownsamples can now be interpolated from the calibration curve generatedwith a reference standard. The major allergen content is determinedrelative to the reference standard (see e.g Allergy Methods andProtocols, Jones and Lympany (2008), p. 138-141, p. 152-153 and p.159-161).

Different sandwich-ELISA (enzyme linked immunosorbent assay) methods canbe used for determination of the major allergen content in Der p and Derfar. One for determination of Der f 1, one for determination of Der p 1and one for determination of Der group 2, since the two group 2 majorallergens are immunochemically identical.

Monoclonal antibodies specific for the major allergen (Der f 1, Der p 1,Der group 2) are adsorbed to the wells of a 96-well microtitre plate.The mAbs recognize epitopes on the major allergen and bind the allergenpresent in the sample. The bound antibody-antigen is washed and aperoxidase labelled rabbit anti-major allergen IgG antibody is added. Byaddition of enzyme substrate a colour reaction will develop, which isdirectly proportional to the content of bound major allergen. A standardcurve is also analyzed in the analytical run and the major allergenactivity is interpolated on the calibration curve. Further to the use ofan internal standard, Center for Biologics Evaluation and Research underFDA use a competition ELISA as described in “Potency Limits forStandardized Dust Mite and Grass Allergen Vaccines: A revised protocol.2000(http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Allergenics/ucm071931.htm)for the potency labelling of allergen products.

In another preferred embodiment the concentration of group 1 and group 2allergen is measured by MS (mass spectrometry) as described in WO2007/031080 using the following calibration standard peptides:

SEQ ID NO: 1 TNACSINGNAPAEIDLR (Der p 1), SEQ ID NO: 2NSWDTTWGDSGYGYFQAGNNLMMIEQYPYVVIM (Der f 1), SEQ ID NO: 3VLVPGCHGSEPCIIHR (Der p 2). SEQ ID NO: 4 GKPFTLEALFDANQNTK (Der f 2),and SEQ ID NO: 5 GIEYIQQNGVVEER (Der f 1)

When the concentration of group 1 and group 2 allergen has beendetermined for a number of mite extracts, mite body extracts and faecalparticles extracts are mixed to achieve the desired weight ratio betweengroup 1 and group 2 allergen.

Determining which extract and the weight of said extracts to be mixed isan iterative process where the amounts by weight of the extracts fromdifferent batches to be used is determined and filled in the followingformula for Der f until the desired ratio is reached:

Major allergen ratio=Total group 1/Total group2=mf1×Cf1grp1+mb1×Cb1grp1+mf2×Cf2grp1+ . . ./mf1×Cf1grp2+mb1×Cb1grp2+mf2×Cf2grp2+ . . .

Where mf1 is mass of the first Der f faecal particles extract used, mf2is mass of the second Der f faecal extract used etc. . . . . Cf1grp1 isconcentration of group 1 allergen in mf1, Cf2grp1 is the concentrationof group 1 allergen the second faecal extracts used, mb1 is the firstmite body extract used and Cb1grp1 is the concentration of group 1allergen in mb1. Cf1grp2 is the concentration of group 2 allergen inmf1, Cb1grp2 is allergen concentration in mb1 and Cf2grp2 is theconcentration group 2 allergen in mf2 etc

A similar formula for Der p is used to determine which and how muchextract to mix to form an extract with the desired ratio of group 1 togroup 2 allergen.

The individual extracts are thawed and mixed according to the formulaabove.

The mixture of allergen extracts may be stabilized and stored as frozendroplets as described in WO 05/0058474 or used directly in aformulation.

The extracts are normally subjected to other standardization processesinvolving a number of tests, e.g. a potency test, before they arerelease for formulation.

The mixture of mite allergen extracts may be use as the activeingredient in a solid dose formulation for sublingual administration asdescribed in WO 04/77994.

Alternatively, the mixture of allergen extracts may be formulated as aliquid formulation for sublingual administration as drops. Suchformulation typically comprises about 50% glycerol, NaCl and NaOH.

The mixture may also be formulated as a liquid formulation forsubcutaneous injection. The extract may either be administered as anaqueous liquid formulation without adjuvants or with an adjuvant such asaluminum hydroxide.

Allergoids may be prepared by cross-linking extracts with across-linking agent which is typically an aldehyde, such asglutaraldehyde. Processes for preparation allergoids are known in theart.

Further Embodiments According to the Invention

Embodiment 1. A pharmaceutical product comprising an allergen extract oran allergoid thereof, which comprises at least one extract of mitebodies selected from the following groups a)-b):

-   -   a) An extract of Der p mite bodies,    -   b) An extract of Der f mite bodies,    -   and at least one extract of mite cultures selected from the        following groups c)-g):    -   c) An extract of Der p faecal particles,    -   d) An extract of Der f faecal particles,    -   e) An extract of Der f whole mite culture,    -   f) An extract of an Der p whole mite culture,    -   g) a combination of extracts c) to f).

Embodiment 2. A pharmaceutical product comprising an allergen extract oran allergoid thereof, which comprises at least one extract of mitebodies selected from the following groups a)-b):

-   -   a) An extract of Der p mite bodies,    -   b) An extract of Der f mite bodies,    -   and at least one extract of mite faecal particles selected from        the following groups c)-d):    -   c) An extract of Der p faecal particles,    -   d) An extract of Der f faecal particles.

Embodiment 3. The pharmaceutical product according to any one ofembodiments 1-2,

wherein the extract of Der p mite bodies is prepared from one fractionor combined fraction(s) of mite cultures said fraction(s) comprisingmore than 70% v/v Der p mite bodies, more preferred more than 75% v/vDer p mite bodies, more preferred more than 80% v/v Der p mite bodies,preferably more than 85% v/v Der p mite bodies, preferably more than 90%v/v Der p mite bodies, preferred more than 95% v/v Der p mite bodies andmost preferred more than 98% v/v Der p mite bodies, and/or

the extract of Der f mite bodies is prepared from one fraction orcombined fraction(s) of mite cultures said fractions comprising morethan 70% v/v Der f mite bodies, preferably more than 75% v/v Der f mitebodies, preferably more than 80% v/v Der f mite bodies, preferably morethan 85% v/v Der f mite bodies, preferably more than 90% v/v Der f mitebodies, more preferred more than 95% v/v Der f mite bodies, andpreferably more than 98% v/v Der f mite bodies.

Embodiment 4. A pharmaceutical product comprising allergen extract,which comprises allergens selected from Der f 1, Der f 2, Der p 1 andDer p 2 and where the allergen present in the lowest concentration (w/v)in the allergen extract, is present in a concentration which is above50% of the concentration of the allergen selected from Der f 1, Der f 2,Der p 1 and Der p 2 present in the highest concentration (w/v), morepreferred above 60% of the concentration of the allergen selected fromDer f 1, Der f 2, Der p 1 and Der p 2 present in the highestconcentration (w/v), more preferred above 70% of the concentration ofthe allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2 presentin the highest concentration (w/v), more preferred above 80% of theconcentration of the allergen selected from Der f 1, Der f 2, Der p 1and Der p 2 present in the highest concentration (w/v), more preferredabove 85% of the concentration of the allergen selected from Der f 1,Der f 2, Der p 1 and Der p 2 present in the highest concentration (w/v),more preferred above 90% of the concentration of the allergen selectedfrom Der f 1, Der f 2, Der p 1 and Der p 2 present in the highestconcentration (w/v), more preferred above 95% of the concentration ofthe allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2 presentin the highest concentration (w/v), preferred above 98% of theconcentration of the allergen selected from Der f 1, Der f 2, Der p 1and Der p 2 present in the highest concentration (w/v), or mostpreferred above 99% of the concentration of the allergen selected fromDer f 1, Der f 2, Der p 1 and Der p 2 present in the highestconcentration (w/v).

Embodiment 5. The pharmaceutical product according to any one ofembodiments 1-4 comprising an extract of Der p mite bodies and anextract of Der p faecal particles.

Embodiment 6. The pharmaceutical product according to any one ofembodiments 1-4 comprising an extract of Der f mite bodies and anextract of Der f faecal particles.

Embodiment 7. The pharmaceutical product according to any one ofembodiments 1-4 comprising an extract of Der p mite bodies and anextract of Der f mite bodies.

Embodiment 8. The pharmaceutical product according any one ofembodiments 1-7 comprising an extract of Der p mite bodies, an extractof Der p faecal particles, an extract of Der f mite bodies and anextract of Der f 2 faecal particles.

Embodiment 9. The pharmaceutical product according to any one ofembodiments 4-8, wherein the extract of Der p mite bodies is preparedfrom one fraction or combined fraction(s) of mite cultures saidfraction(s) comprising more than 70% v/v Der p mite bodies, morepreferred more than 75% v/v Der p mite bodies, more preferred more than80% v/v Der p mite bodies, preferably more than 85% v/v Der p mitebodies, preferably more than 90% v/v Der p mite bodies, preferred morethan 95% v/v Der p mite bodies and most preferred more than 98% v/v Derp mite bodies, and/or

the extract of Der f mite bodies is prepared from one fraction orcombined fraction(s) of mite cultures said fractions comprising morethan 70% v/v Der f mite bodies, preferably more than 75% v/v Der f mitebodies, preferably more than 80% v/v Der f mite bodies, preferably morethan 85% v/v Der f mite bodies, preferably more than 90% v/v Der f mitebodies, more preferred more than 95% v/v Der f mite bodies, andpreferably more than 98% v/v Der f mite bodies.

Embodiment 10. The pharmaceutical product according to any one ofembodiments 1-9, wherein the extract of Der p mite bodies is preparedfrom one Der p mite body fraction or combined Der p mite body fractionsand where extraction of said fraction(s) of mite bodies results in anextract comprising by weight equal amounts of group 1 and group 2allergen or more group 2 allergen than group 1 allergen and/or theextract of Der f mite bodies is prepared from one Der f mite bodyfraction or more combined Der f mite body fractions and where extractionof said fraction(s) of mite bodies results in an extract comprising byweight equal amounts by weight of group 1 and group 2 allergen or moregroup 2 allergen than group 1 allergen.

Embodiment 11. The pharmaceutical product according to any one ofembodiments 1-10, wherein the extract of Der p mite bodies is preparedfrom one fraction or combined fraction(s) of mite cultures saidfraction(s) comprising more than 70% v/v Der p mite bodies, morepreferred more than 75% v/v Der p mite bodies, more preferred more than80% v/v Der p mite bodies, preferably more than 85% v/v Der p mitebodies, preferably more than 90% v/v Der p mite bodies, preferred morethan 95% v/v Der p mite bodies and most preferred more than 98% v/v Derp mite bodies, and/or

the extract of Der f mite bodies is prepared from one fraction orcombined fraction(s) of mite cultures said fractions comprising morethan 70% v/v Der f mite bodies, preferably more than 75% v/v Der f mitebodies, preferably more than 80% v/v Der f mite bodies, preferably morethan 85% v/v Der f mite bodies, preferably more than 90% v/v Der f mitebodies, more preferred more than 95% v/v Der f mite bodies, andpreferably more than 98% v/v Der f mite bodies.

Embodiment 12. The pharmaceutical product according to any one ofembodiments 1-11, wherein

the extract of Der p faecal particles is prepared from one fraction orcombined fraction(s) of mite cultures said fraction(s) comprising morethan 70% v/v Der p faecal particles, preferably more than 75% v/v Der pfaecal particles, preferably more than 80% v/v Der p faecal particles,preferably more than 85% v/v Der p faecal particles, preferably morethan 90% v/v Der p faecal particles, more preferred more than 95% v/vDer p faecal particles, most preferred more than 98% v/v Der p faecalparticles and/or

the extract of Der f faecal particles is prepared from one fraction orcombined fraction(s) of mite cultures said fraction(s) comprising morethan 70% v/v Der f faecal particles, preferred more than 75% v/v Der ffaecal particles, preferred more than 80% v/v Der f faecal particles,preferably more than 85% v/v Der f faecal particles, preferably morethan 90% v/v Der f faecal particles, preferred more than 95% v/v, morepreferred more than 98% v/v Der f faecal particles.

Embodiment 13. The pharmaceutical product according to embodiment 12,wherein a fraction or the combined fractions of mite bodies and/orwherein a fraction or the combined fractions of mite faecal particleseach comprises below 10% v/v of mite culture medium.

Embodiment 14. The pharmaceutical product according to embodiment 13,wherein the mite body fraction or combined body fractions comprises upto 20% v/v mite parts (e.g. legs), faecal particles and mite medium.

Embodiment 15. The pharmaceutical product according to any one ofembodiments 12-14, wherein the mite faecal fraction or combined faecalfractions comprises up to 20% v/v mite parts (e.g. legs) and mitemedium.

Embodiment 16. The pharmaceutical product according to any one ofembodiments 1-15, wherein the amount by weight of group 1 allergen islower than the amount by weight of group 2 allergen in the extracts ofmite body and the amount by weight of group 1 allergen is higher thanthe amount by weight of group 2 allergen in the extracts of mite faecalparticles.

Embodiment 17. The pharmaceutical product according to any one ofembodiments 1-16 comprising allergen extract, which comprises allergensselected from Der f 1, Der f 2, Der p 1 and Der p 2 and where theallergen present in the lowest concentration (w/v) in the allergenextract, is present in a concentration which is above 50% of theconcentration of the allergen selected from Der f 1, Der f 2, Der p 1and Der p 2 present in the highest concentration (w/v), more preferredabove 60% of the concentration of the allergen selected from Der f 1,Der f 2, Der p 1 and Der p 2 present in the highest concentration (w/v),more preferred above 70% of the concentration of the allergen selectedfrom Der f 1, Der f 2, Der p 1 and Der p 2 present in the highestconcentration (w/v), more preferred above 80% of the concentration ofthe allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2 presentin the highest concentration (w/v), more preferred above 85% of theconcentration of the allergen selected from Der f 1, Der f 2, Der p 1and Der p 2 present in the highest concentration (w/v), more preferredabove 90% of the concentration of the allergen selected from Der f 1,Der f 2, Der p 1 and Der p 2 present in the highest concentration (w/v),more preferred above 95% of the concentration of the allergen selectedfrom Der f 1, Der f 2, Der p 1 and Der p 2 present in the highestconcentration (w/v), preferred above 98% of the concentration of theallergen selected from Der f 1, Der f 2, Der p 1 and Der p 2 present inthe highest concentration (w/v), or most preferred above 99% of theconcentration of the allergen selected from Der f 1, Der f 2, Der p 1and Der p 2 present in the highest concentration (w/v).

Embodiment 18. The pharmaceutical product according to any one ofembodiments 1-17, wherein the pharmaceutical product is a compressed ornon-compressed sublingual tablet(s) comprising the extract(s) orallergoids prepared from an extract(s), a liquid sublingual productcomprising the extract(s) or allergoids prepared from extract(s), or aliquid product for injection comprising the extract(s) or allergoidsprepared from extract(s).

Embodiment 19. The pharmaceutical product according to embodiment 18,wherein the pharmaceutical product is a fast dissolving sublingualtablet which comprises an extract.

Embodiment 20. The pharmaceutical product according to any of theembodiments 1-19, further comprising an extract of a whole mite cultureor a body fraction(s) having less than 70% v/v of mite bodies.

Embodiment 21. The pharmaceutical product according to any of theembodiments 1-20 for the treatment and/or prevention of allergy andallergic asthma caused by house dust mites and/or for the diagnosis ofallergy.

Embodiment 22. Use of the pharmaceutical product according to any of theembodiments 1-21, for the preparation of a medical product for thetreatment and/or prevention of allergy and allergic asthma caused byhouse dust mites and/or for the diagnosis of allergy.

Embodiment 23. A mite body fraction comprising more than 70% v/v mitebodies, more preferred more than 75% v/v mite bodies, more preferredmore than 80% v/v mite bodies, preferably more than 85% v/v mite bodies,preferably more than 90% v/v mite bodies, preferred more than 95% v/vmite bodies and most preferred more than 98% v/v mite bodies.

Embodiment 24. The mite body fraction according to embodiment 23,wherein the mite bodies are Der p mite bodies or Der f mite bodies.

Embodiment 25. The mite body fraction according to any one ofembodiments 23-24 containing below 10% v/v culture media particles.

Embodiment 26. The mite body fraction according to any one ofembodiments 23-25 comprising less than 20% v/v mite parts, faecalparticles and mite medium.

Embodiment 27. An extract of a mite body fraction as defined in any oneof embodiments 23-26 for use in a pharmaceutical product for thetreatment and/or prevention of allergy and allergic asthma caused byhouse dust mites.

Embodiment 28. A mite faecal particle fraction comprising more than 70%v/v faecal particles, preferably more than 75% v/v faecal particles,preferably more than 80% v/v faecal particles, preferably more than 85%v/v faecal particles, preferably more than 90% v/v faecal particles,more preferred more than 95% v/v faecal particles, most preferred morethan 98% v/v faecal particles.

Embodiment 29. The mite faecal particle fraction according to embodiment28, wherein the faecal particles is Der p faecal particles or Der ffaecal particles.

Embodiment 30. The mite faecal particle fraction according to any one ofembodiments 28-29 comprising up to 20% v/v mite parts (e.g. legs) andmite medium.

Embodiment 31. An extract of a mite faecal particle fraction as definedin any one of embodiments 28-30 for use in a pharmaceutical product forthe treatment and/or prevention of allergy and allergic asthma caused byhouse dust mites.

Embodiment 32. Use of an extract according to any one of embodiments 27and 31 for the preparation of a pharmaceutical product for use in apharmaceutical product for the treatment and/or prevention of allergyand allergic asthma caused by house dust mites.

Embodiment 33. A method for the manufacture of a mite allergen extractcomprising Der p 1 and Der p 2 allergen, which method comprises thefollowing steps:

-   -   a. Isolating fraction(s) of Der p mite bodies from cultures of        Der p mites, where said fraction(s) comprises more than 70% v/v        Der p mite bodies, more preferred more than 75% v/v Der p mite        bodies, more preferred more than 80% v/v Der p mite bodies,        preferably more than 85% v/v Der p mite bodies, preferably more        than 90% v/v Der p mite bodies, preferred more than 95% v/v Der        p mite bodies and most preferred more than 98% v/v Der p mite        bodies;    -   b. Optionally combining several of said fraction(s) of Der p        mite bodies; and    -   c. Extracting allergens from said Der p mite body fraction(s)        obtained in a step a) or a step b) as above to obtain said mite        allergen extract.

Embodiment 34. A method for the manufacture of a mite allergen extractcomprising Der f 1 and Der f 2 allergens, which method comprises thefollowing steps:

-   -   a. Isolating fraction(s) of Der f mite bodies from cultures of        Der f mites, where said fraction(s) comprises more than 70% v/v        Der f mite bodies, more preferred more than 75% v/v Der f mite        bodies, more preferred more than 80% v/v Der f mite bodies,        preferably more than 85% v/v Der f mite bodies, preferably more        than 90% v/v Der f mite bodies, preferred more than 95% v/v Der        f mite bodies and most preferred more than 98% v/v Der f mite        bodies;    -   b. Optionally combining several of said fraction(s) of Der f        mite bodies; and    -   c. Extracting allergens from said Der f mite body fraction(s)        obtained in a step a) or a step b) as above to obtain said mite        allergen extract.

Embodiment 35. The method according any one of embodiments 33-34, whichmethod further comprises the following step: mixing one or more miteallergen extract(s) of embodiment 33 with one or more mite allergenextract(s) of embodiment 34 to obtain a mixture of mite allergenextracts comprising Der p 1, Der p 2, Der f 1, and Der f 2 allergen.

Embodiment 36. The method according any one of embodiments 33-35, whichmethod further comprises the following steps:

-   -   a. Isolating fraction(s) of Der p mite faecal particles from        cultures of Der p mites;    -   b. Optionally combining several of said fraction(s) of Der p        mite faecal particles;    -   c. Extracting allergens from Der p mite faecal particle        fraction(s) obtained in a step a) or a step b) as above; and    -   d. Mixing one or more mite allergen extract(s) of any one of        embodiments 33-35 with one or more extract(s) of faecal        particles as obtained in a step c) above to obtain a mixture of        mite allergen extracts.

Embodiment 37. The method according to any one of embodiments 33-36,which method further comprises the following steps:

-   -   a. Isolating fraction(s) of Der f mite faecal particles from        cultures of Der f mites;    -   b. Optionally combining several of said fraction(s) of Der f        mite faecal particles;    -   c. Extracting allergens from Der f mite faecal particle        fraction(s) obtained in a step a) or a step b) as above; and    -   d. Mixing one or more mite allergen extract(s) of any one of        embodiments 33-35 with one or more extract(s) of faecal        particles as obtained in a step c) above to obtain a mixture of        mite allergen extracts.

Embodiment 38. The method according to any one of embodiments 33-37,which method further comprises the following step: mixing one or moremite allergen extract(s) of embodiment 33 and embodiment 34 with one ormore mite allergen extract(s) of step c in embodiment 36 and of step cin embodiment 37 to obtain a mixture of mite allergen extractscomprising Der p 1, Der p 2, Der f 1, and Der f 2 allergens.

Embodiment 39. The method according to any one of embodiments 33-38,wherein the obtained mite allergen extract is converted to an allergoidthereof.

Embodiment 40. The method according to any one of embodiments 33-39,which method comprises a further step of

-   -   a. Measuring the concentration (w/v) of group 1 and group 2        allergen in mite allergen extracts of step c) of any one of        embodiments 33-34 and 36-37.

Embodiment 41. The method according to embodiment 40, which methodcomprises a further step of

-   -   e) Mixing one or more extract(s) of mite bodies of a step c) of        any one of embodiments 33-34 with one or more extract(s) of        faecal particles of a step c) of any one of embodiments 36-37 to        obtain a mixture of mite allergen extracts with a predetermined        amount by weight of group 1 to group 2 allergen.

Embodiment 42. The method according to any one of embodiments 33-41,wherein the mixture of mite allergen extract comprises extract(s) of Derp mite bodies, extract(s) of Der p faecal particles, extract(s) of Der fmite bodies and extract(s) of Der f 2 faecal particles.

Embodiment 43. The method according to any one of embodiments 33-42,wherein more than one fractions of mite bodies are combined according tostep b) followed by extraction according to step c) of embodiment 33and/or 34.

Embodiment 44. The method according to any one of embodiments 33-43,wherein more than one fraction of faecal particles are combinedaccording to step b) followed by extraction according to step c) ofembodiment 36 and/or 37.

Embodiment 45. The method according to any one of embodiments 33-44,wherein one or more extract(s) of mite bodies obtained in a step c) ofembodiment 33 and/or 34 is mixed in a step e) with one or moreextract(s) of mite faecal particles obtained in a step c) of embodiment36 and/or 37.

Embodiment 46. The method according to any one of embodiments 33-45,wherein one or more extract(s) of mite bodies obtained in a step c) ofembodiment 33 and/or 34 is mixed in a step e) with one extract of mitefaecal particles obtained in a step c) of embodiment 36 and/or 37.

Embodiment 47. The method according to any one of embodiments 33-46,wherein one extract of mite bodies obtained in a step c) of embodiment33 and/or 34 is mixed in a step with one or more extract(s) of mitefaecal particles obtained in a step c) of embodiment 36 and/or 37.

Embodiment 48. The method according to any one embodiments 33-47,wherein a mite body extract(s) is supplemented with an amount by weightof faecal particles extract, a body extract from a body fraction of lessthan 70% v/v or a whole culture extract, which is sufficient to achievea desired group 1 allergen to group 2 weight ratio in the mixture ofmite allergen extracts.

Embodiment 49. The method according to any one of embodiments 33-48,wherein

the extract of Der p faecal particles in step c) of embodiment 36 isprepared from one fraction or combined fraction(s) of mite cultures saidfraction(s) comprising more than 70% v/v Der p faecal particles,preferably more than 75% v/v Der p faecal particles, preferably morethan 80% v/v Der p faecal particles, preferably more than 85% v/v Der pfaecal particles, preferably more than 90% v/v Der p faecal particles,more preferred more than 95% v/v Der p faecal particles, most preferredmore than 98% v/v Der p faecal particles and/or

the extract of Der f faecal particles in step c) of embodiment 37 isprepared from one fraction or combined fraction(s) of mite cultures saidfraction(s) comprising more than 70% v/v Der f faecal particles,preferred more than 75% v/v Der f faecal particles, preferred more than80% v/v Der f faecal particles, preferably more than 85% v/v Der ffaecal particles, preferably more than 90% v/v Der f faecal particles,preferred more than 95% v/v, more preferred more than 98% v/v Der ffaecal particles.

Embodiment 50. The method according to any one of embodiments 33-49,wherein a fraction of mite bodies in step a) or the combined fractionsof mite bodies in step b) of embodiment 33 and/or 34 and/or wherein afraction of faecal particles in step a) or the combined fractions ofmite faecal particles in step b) of embodiment 36 and/or 37 eachcomprises below 10% v/v of mite culture medium.

Embodiment 51. The method according to embodiment 50, wherein the mitebody fraction in step a) or the combined body fractions in step b) ofembodiment 33 and/or 34 comprises up to 20% v/v mite parts (e.g. legs),faecal particles and mite medium.

Embodiment 52. The method according to embodiment 50, wherein the mitefaecal fraction in step a) or combined faecal fractions in step b) ofembodiment 36 and/or 37 comprises up to 20% v/v mite parts (e.g. legs)and mite medium.

Embodiment 53. The method according to any one of embodiments 33-52,wherein the fraction(s) of mite bodies of embodiment 33 and/or 34 andthe fraction(s) of mite faecal particles in step a) of embodiment 36and/or 37 is isolated from mite culture(s) by sieving of the Der pand/or Der f mite culture(s) to produce a mite body fraction b) and amite faecal particles fraction d).

Embodiment 54. The method according to embodiment 33-53, wherein thefraction(s) of mite bodies and the fraction(s) of mite faecal particlesis obtained by sieving of the Der p and/or Der f mite culture(s) toproduce four fractions:

a. Large medium particles >350 μm

b. Mite body fraction 350-90 μm

c. Faecal agglomerates and mite parts 90-50 μm

d. Faecal particles <50 μm

e. fraction a) and c) are discarded, fraction d) and b) are optionallysubjected to further purification or used directedly for extraction.

Embodiment 55. The method according to embodiment 54, wherein mite bodyfraction b) is:

-   -   ii) Subjected to density centrifugation in a first liquid medium        having a higher density than the mite bodies and a lower density        than the culture media particles; followed by    -   iii) Collection of the mite bodies from said first liquid medium        and optionally repeating step ii) and iii) until the desired        purity of the mite bodies have been achieved, optionally        followed by    -   iv) Washing of the mite bodies to remove said first liquid with        a second liquid.

Embodiment 56. A method for the isolation and purification of mitebodies comprising the steps:

-   -   i) Isolating mite body fraction(s) from Der p or Der f mite        culture(s) and    -   ii) Subjecting one or more Der p mite body fraction(s), or one        or more Der f mite body fraction(s), to density centrifugation        in a first liquid medium having a higher density than the mite        bodies and a lower density than the culture media particles; and    -   iii) Collecting the mite bodies from said first liquid medium    -   and optionally repeating step ii) and iii) until the desired        purity of the mite bodies have been achieved, optionally        followed by    -   iv) Washing of the mite bodies to remove said first liquid with        a second liquid.

Embodiment 57. A method for the isolation and purification of mitebodies comprising the steps:

-   -   i) Isolating mite body fraction(s) from Der p or Der f mite        culture(s) and    -   ii) Subjecting one or more Der p mite body fraction(s), or one        or more Der f mite body fraction(s), to density centrifugation        in a first liquid medium having a higher density than the mite        bodies and a lower density than the culture media particles; and    -   iii) Collecting the mite bodies from said first liquid medium,        optionally followed by    -   iv) Washing of the mite bodies to remove said first liquid with        a second liquid.

Embodiment 58. The method according to any one of embodiments 56-57,wherein the first and second liquids are anhydrous, preferablypharmaceutically acceptable liquids.

Embodiment 59. The method according to any one of embodiments 56-58,wherein the first liquid is anhydrous glycerol.

Embodiment 60. The method according to any one of embodiments 56-59,wherein the second liquid is anhydrous ethanol.

Embodiment 61. The method according to any one of embodiments 56-60,wherein step ii) and iii) is repeated once.

Embodiment 62. The method according to any one of embodiments 56-61,wherein step ii) and iii) are repeated until the mite body fractionobtained comprises at least 70% v/v mite bodies, more preferred morethan 75% v/v mite bodies, more preferred more than 80 ° A) v/v mitebodies, preferably more than 85% v/v mite bodies, preferably more than90% v/v mite bodies, preferred more than 95% v/v mite bodies and mostpreferred more than 98% v/v mite bodies.

Embodiment 63. The method of embodiment 56-62, wherein purification ofthe mite body fraction is continued until the mite body fraction containbelow 10% v/v culture media particles.

Embodiment 64. The method of embodiment 63, wherein the purification ofthe mite body is continued until less than 20% v/v mite parts, faecalparticles and mite medium is present.

Embodiment 65. The method of any one of embodiments 33-64, whereinfractions of Der f mite bodies, fractions of Der p mite bodies,fraction(s) of Der p faecal particles and/or fractions of Der f faecalparticles in step a) of any one of embodiment 33-34 and 36-37 isisolated by a sieving process comprising:

-   -   1) Sieving Der f or Der p mite cultures in a vibrating sieve        shaker with a tower of a 300, a 150 and a 80 μm mesh sieve        followed by isolation of a mite body rich fraction between 150        μm and 300 μm and a mite faecal particles rich fraction below 80        μm, followed by    -   2) Sieving the mite body rich fraction (150 μm and 300 μm) in a        sieve shaker with a horizontally and circular movement and with        a tower of 200, 300 and/or 400 μm mesh sieves and isolating body        fraction(s) captured by the 200, 300 and/or 400 μm mesh sieves,        followed by    -   3) Sieving of the body rich fraction in a sieve shaker having a        3D throwing motion and 300, 200, 150 and 100 mesh sieve to        produce a mite body fraction between 150 and 300 μm mesh and a        mite faecal particles extract below 100 μm; and    -   4) Optionally combining the facal particles fraction below 80 μm        from the vibrating sieve and the faecal particles fraction below        100 μm from the sieve with the 3D throwing motion and subjecting        the combined faecal fraction to sieving in the sieve with the 3D        throwing motion and a tower of 100, 80, 50 μm mesh sieves and        isolating the faecal particles rich fraction blow 50 μm.

Embodiment 66. The method according to any one of embodiments 33-65,wherein the extracts or mixture of extracts obtained are formulated in apharmaceutical product such as a compressed or non-compressed sublingualtablet(s) comprising the extract(s) or allergoids prepared from anextract(s), a liquid sublingual product comprising the extract(s) orallergoids prepared from extract(s), or a liquid product for injectioncomprising the extract(s) or allergoids prepared from extract(s).

Embodiment 67. The method according to embodiment 66, wherein thepharmaceutical product is a fast dissolving sublingual tablet whichcomprises an extract.

Embodiment 68. A pharmaceutical product prepared by a method as definedin any one of the embodiments 33-67.

Embodiment 69. The pharmaceutical product according to embodiment 68 forthe treatment and/or prevention of allergy and allergic asthma caused byhouse dust mites.

Embodiment 70. An allergen extract of a series of allergen extracts forthe use in a pharmaceutical allergen product for the treatment and/orprevention of allergy and allergic asthma caused by house dust mites,each allergen extract comprising at least one extract of mite bodies,and at least one extract of mite faecal particles, or a mite bodyextract from a fraction of mite bodies having less than 70% v/v mitebodies or an whole mite culture extract, wherein said allergen extracthas a predetermined ratio of group 1 and group 2 allergen, and whereinthe variance coefficient of said ratio is no more than 25, 20, 15, 10,or 7.5% for the series of extracts.

Embodiment 71. An allergen extract of an series of allergen extracts forthe use in a pharmaceutical allergen product for the diagnosis ofallergy caused by house dust mites, each allergen extract comprising atleast one extract of mite bodies and at least one extract of mite faecalparticles, or a mite body extract from a fraction of mite bodies havingless than 70% v/v mite bodies or an whole mite culture extract, whereinsaid allergen extract has a predetermined ratio of group 1 and group 2allergen, and wherein the variance coefficient of said ratio is no morethan 25, 20, 15, 10, or 7.5% for the series of extracts.

Embodiment 72. The allergen extract according to any one of embodiments70-71, wherein the amount by weight of group 1 allergen is lower thanthe amount by weight of group 2 allergen in the extracts of mite bodyand the amount by weight of group 1 allergen is higher than the amountby weight of group 2 allergen in the extracts of mite faecal particles.

Embodiment 73. The allergen extract according to any one of embodiments70-72, wherein the allergen extract is as defined in any one ofembodiments 1-32.

Embodiment 74. The allergen extract according to any one of embodiments70-72, wherein the allergen extract is prepared as defined in any one ofembodiments 33-66.

Further Embodiments According to the Invention

Embodiment 1. A pharmaceutical product comprising an allergen extract(I) or an allergoid thereof for the treatment and/or prevention ofallergy and allergic asthma caused by house dust mites comprising atleast one extract of mite bodies and optionally at least one extract ofmite faecal particles selected from the following groups a)-d):

-   -   a) An extract of Der p mite bodies,    -   b) An extract of Der p faecal particles,    -   c) An extract of Der f mite bodies, and    -   d) An extract of Der f faecal particles.

Embodiment 2. A pharmaceutical product comprising allergen extract (I)for the treatment and/or prevention of allergy and allergic asthmacaused by house dust mites comprising allergens selected from Der f 1,Der f 2, Der p 1 and Der p 2 and where the allergen present in thelowest concentration (w/v) in the allergen extract (I), is present in aconcentration which is above 50% of the concentration of the allergenselected from Der f 1, Der f 2, Der p 1 and Der p 2 present in thehighest concentration (w/v), more preferred above 60% of theconcentration of the allergen selected from Der f 1, Der f 2, Der p 1and Der p 2 present in the highest concentration (w/v), more preferredabove 70% of the concentration of the allergen selected from Der f 1,Der f 2, Der p 1 and Der p 2 present in the highest concentration (w/v),more preferred above 80% of the concentration of the allergen selectedfrom Der f 1, Der f 2, Der p 1 and Der p 2 present in the highestconcentration (w/v), more preferred above 85% of the concentration ofthe allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2 presentin the highest concentration (w/v), more preferred above 90% of theconcentration of the allergen selected from Der f 1, Der f 2, Der p 1and Der p 2 present in the highest concentration (w/v), more preferredabove 95% of the concentration of the allergen selected from Der f 1,Der f 2, Der p 1 and Der p 2 present in the highest concentration (w/v),preferred above 98 ° A) of the concentration of the allergen selectedfrom Der f 1, Der f 2, Der p 1 and Der p 2 present in the highestconcentration (w/v), or most preferred above 99% of the concentration ofthe allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2 presentin the highest concentration (w/v).

Embodiment 3. A pharmaceutical product according to any of embodiments1-2 comprising at least one extract of mite bodies and at least oneextract of mite faecal particles.

Embodiment 4. The pharmaceutical product according to embodiment 3comprising an extract of Der p mite bodies, an extract of Der p faecalparticles, an extract of Der f mite bodies and an extract of Der f 2faecal particles.

Embodiment 5. A pharmaceutical product according to any of embodiments1-2 comprising an extract of Der p mite bodies and an extract of Der pfaecal particles and no extracts of Der f mite bodies and no extracts ofDer f faecal particles.

Embodiment 6. A pharmaceutical product according to any of embodiments1-2 comprising an extract of Der f mite bodies and an extract of Der ffaecal particles and no extracts of Der p mite bodies and no extracts ofDer p faecal particles.

Embodiment 7. A pharmaceutical product according to any of embodiments1-2 comprising an extract of Der p mite bodies and an extract of Der fmite bodies and no extract of mite faecal particles.

Embodiment 8. A pharmaceutical product according to any of embodiments1-2 comprising an extract of Der p mite bodies and no extract of Der fmite bodies and no extract of mite faecal particles.

Embodiment 9. A pharmaceutical product according to any of embodiment1-2 comprising an extract of Der f mite bodies and no an extract of Derp mite bodies extract and no extract of mite faecal particles.

Embodiment 10. A pharmaceutical product according to any of embodiments1-9 wherein

the extract of Der p mite bodies is prepared from one Der p mite bodyfraction or combined Der p mite body fractions and where extraction ofsaid fraction(s) of mite bodies results in an extract comprising byweight equal amounts of group 1 and group 2 allergen or more group 2allergen than group 1 allergen and/or

the extract of Der f mite bodies is prepared from one Der f mite bodyfraction or more combined Der f mite body fractions and where extractionof said fraction(s) of mite bodies results in an extract comprising byweight equal amounts by weight of group 1 and group 2 allergen or moregroup 2 allergen than group 1 allergen.

Embodiment 11. A pharmaceutical product according to any of embodiments1-10 wherein

the extract of Der p mite bodies is prepared from one fraction orcombined fraction(s) of mite cultures said fraction(s) comprising morethan 70% v/v Der p mite bodies, more preferred more than 75% v/v Der pmite bodies, more preferred more than 80% v/v Der p mite bodies,preferably more than 85% v/v Der p mite bodies, preferably more than 90%v/v Der p mite bodies, preferred more than 95% v/v Der p mite bodies andmost preferred more than 98% v/v Der p mite bodies,

the extract of Der f mite bodies is prepared from one fraction orcombined fraction(s) of mite cultures said fractions comprising morethan 70% v/v Der f mite bodies, preferably more than 75% v/v Der f mitebodies, preferably more than 80% v/v Der f mite bodies, preferably morethan 85% v/v Der f mite bodies, preferably more than 90% v/v Der f mitebodies, more preferred more than 95% v/v Der f mite bodies, andpreferably more than 98% v/v Der f mite bodies,

the extract of Der p faecal particles is prepared from one fraction orcombined fraction(s) of mite cultures said fraction(s) comprising morethan 70% v/v Der p faecal particles, preferably more than 75% v/v Der pfaecal particles, preferably more than 80% v/v Der p faecal particles,preferably more than 85% v/v Der p faecal particles, preferably morethan 90% v/v Der p faecal particles, more preferred more than 95% v/vDer p faecal particles, most preferred more than 98% v/v Der p faecalparticles and/or

the extract of Der f faecal particles is prepared from one fraction orcombined fraction(s) of mite cultures said fraction(s) comprising morethan 70% v/v Der f faecal particles, preferred more than 75% v/v Der ffaecal particles, preferred more than 80% v/v Der f faecal particles,preferably more than 85% v/v Der f faecal particles, preferably morethan 90% v/v Der f faecal particles, preferred more than 95% v/v, morepreferred more than 98% v/v Der f faecal particles.

Embodiment 12. The pharmaceutical product according to embodiment 11wherein a fraction or the combined fractions of mite bodies and/orwherein a fraction or the combined fractions of mite faecal particleseach comprises below 10% v/v of mite culture medium.

Embodiment 13. The pharmaceutical product according to embodiment 12wherein the mite body fraction or combined body fractions comprises upto 20% v/v mite parts (e.g. legs), faecal particles and mite medium.

Embodiment 14. The pharmaceutical product according to embodiment 12wherein the mite faecal fraction or combined faecal fractions comprisesup to 20% v/v mite parts (e.g. legs) and mite medium.

Embodiment 15. The pharmaceutical product according to any ofembodiments 1-14 wherein the amount by weight of group 1 allergen islower than the amount by weight of group 2 allergen in the extracts ofmite body and the amount by weight of group 1 allergen is higher thanthe amount by weight of group 2 allergen in the extracts of mite faecalparticles.

Embodiment 16. The pharmaceutical product according to any ofembodiments 1, 3-15 comprising allergen extract (I) for the treatmentand/or prevention of allergy and allergic asthma caused by house dustmites comprising allergens selected from Der f 1, Der f 2, Der p 1 andDer p 2 and where the allergen present in the lowest concentration (w/v)in the allergen extract (I), is present in a concentration which isabove 50% of the concentration of the allergen selected from Der f 1,Der f 2, Der p 1 and Der p 2 present in the highest concentration (w/v),more preferred above 60% of the concentration of the allergen selectedfrom Der f 1, Der f 2, Der p 1 and Der p 2 present in the highestconcentration (w/v), more preferred above 70% of the concentration ofthe allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2 presentin the highest concentration (w/v), more preferred above 80% of theconcentration of the allergen selected from Der f 1, Der f 2, Der p 1and Der p 2 present in the highest concentration (w/v), more preferredabove 85% of the concentration of the allergen selected from Der f 1,Der f 2, Der p 1 and Der p 2 present in the highest concentration (w/v),more preferred above 90% of the concentration of the allergen selectedfrom Der f 1, Der f 2, Der p 1 and Der p 2 present in the highestconcentration (w/v), more preferred above 95% of the concentration ofthe allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2 presentin the highest concentration (w/v), preferred above 98% of theconcentration of the allergen selected from Der f 1, Der f 2, Der p 1and Der p 2 present in the highest concentration (w/v), or mostpreferred above 99% of the concentration of the allergen selected fromDer f 1, Der f 2, Der p 1 and Der p 2 present in the highestconcentration (w/v).

Embodiment 17. The pharmaceutical product according to any ofembodiments 1-16, wherein the pharmaceutical product is a compressed ornon-compressed sublingual tablet(s) comprising the extract (I) orallergoids prepared from an extract (I) in a solid form, a liquidsublingual product or a liquid product for injection comprising theextract (I) or allergoids prepared from extract (I), preferably thepharmaceutical product is a fast dissolving sublingual tablet whichcomprises an extract (I) in solid form.

Embodiment 18. A method for the manufacture of a mite allergen allergenextract (I) or allergoids thereof for a pharmaceutical product accordingto any of embodiments 1-10 and 15-16 said allergen extract (I) having apredetermined and controlled amount by weight of allergens selected fromDer f 1, Der f 2, Der p 1 and Der p 2 allergens and comprising thefollowing steps:

a) Fractions of Der p mite bodies and/or fractions of Der p mite faecalparticles are isolated from Der p cultures of mites, and/or fractions ofDer f mite bodies and/or fractions of Der f mite faecal particles areisolated from Der f mite cultures;

b) Optionally combining several fractions of Der p mite bodies,optionally combining several fractions of Der p mite faecal particles,optionally combining several fractions of Der f mite bodies andoptionally combining several fractions of Der f mite faecal particles;

c) Extracting allergens from mite body faction(s) obtained in a step a)or a step b) as above and/or extraction of allergens from mite faecalparticle fraction(s) obtained in a step a) or a step b) as above; andthereafter

d) Measuring the concentration (w/v) of group 1 and group 2 allergen inmite allergen extracts obtained in a step c) as above; and optionally

e) Mixing one or more extract(s) of mite bodies with one or moreextract(s) of faecal particles as obtained in a step c) as above toobtain a mixture of allergen extracts (I) with a predetermined amount byweight of group 1 to group 2 allergen and optionally

f) Converting the extract (I) to an allergoid thereof.

Embodiment 19. The method according to embodiment 18 wherein one or moreextract(s) of mite bodies and one or more extract(s) of faecal particlesis mixed to obtain a mixture of allergen extracts (I).

Embodiment 20. The method according to embodiments 18-19 wherein themixture of mite allergen extract (I) comprises extract(s) of Der p mitebodies, extract(s) of Der p faecal particles, extract(s) of Der f mitebodies and extract(s) of Der f 2 faecal particles.

Embodiment 21. The method according to any of embodiments 18-20 whereinmore than one fractions of mite bodies are combined according to step b)followed by extraction according to step c).

Embodiment 22. The method according to any of embodiments 18-21 whereinmore than one fraction of faecal particles are combined according tostep b) followed by extraction according to step c).

Embodiment 23. The method according to any of embodiments 18-22 whereinone or more extract(s) of mite bodies obtained in a step c) is mixed ina step e) with one or more extract(s) of mite faecal particles obtainedin a step c).

Embodiment 24. The method according to any of embodiments 18-23 whereinone or more extract(s) of mite bodies obtained in a step c) is mixed ina step e) with one extract of mite faecal particles obtained in a stepc).

Embodiment 25. The method according to any of embodiments 18-24 whereinone extract of mite bodies obtained in a step c) is mixed in a step e)with one or more extract(s) of mite faecal particles obtained in a stepc).

Embodiment 26. The method according to any of embodiment 18-25 wherein amite body extract(s) is supplemented with an amount by weight of faecalparticles extract which is sufficient to achieve the desired group 1allergen to group 2 weight ratio in the mixture of mite allergenextracts (I).

Embodiment 27. A method according to any of embodiments 14-26 wherein

the extract in of Der p mite bodies in step c) is prepared from onefraction or combined fraction(s) of mite cultures said fraction(s)comprising more than 70% v/v Der p mite bodies, more preferred more than75% v/v Der p mite bodies, more preferred more than 80% v/v Der p mitebodies, preferably more than 85% v/v Der p mite bodies, preferably morethan 90% v/v Der p mite bodies, preferred more than 95% v/v Der p mitebodies and most preferred more than 98% v/v Der p mite bodies,

the extract of Der f mite bodies in step c) is prepared from onefraction or combined fraction(s) of mite cultures said fractionscomprising more than 70% v/v Der f mite bodies, preferably more than 75%v/v Der f mite bodies, preferably more than 80% v/v Der f mite bodies,preferably more than 85% v/v Der f mite bodies, preferably more than 90%v/v Der f mite bodies, more preferred more than 95% v/v Der f mitebodies, and preferably more than 98% v/v Der f mite bodies,

the extract of Der p faecal particles in step c) is prepared from onefraction or combined fraction(s) of mite cultures said fraction(s)comprising more than 70% v/v Der p faecal particles, preferably morethan 75% v/v Der p faecal particles, preferably more than 80% v/v Der pfaecal particles, preferably more than 85% v/v Der p faecal particles,preferably more than 90% v/v Der p faecal particles, more preferred morethan 95% v/v Der p faecal particles, most preferred more than 98% v/vDer p faecal particles and/or

the extract of Der f faecal particles in step c) is prepared from onefraction or combined fraction(s) of mite cultures said fraction(s)comprising more than 70% v/v Der f faecal particles, preferred more than75% v/v Der f faecal particles, preferred more than 80% v/v Der f faecalparticles, preferably more than 85% v/v Der f faecal particles,preferably more than 90% v/v Der f faecal particles, preferred more than95% v/v, more preferred more than 98% v/v Der f faecal particles.

Embodiment 28. The method according to embodiment 27 wherein a fractionof mite bodies in step a) or the combined fractions of mite bodies instep b) and/or wherein a fraction of faecal particles in step a) or thecombined fractions of mite faecal particles in step b) each comprisesbelow 10% v/v of mite culture medium.

Embodiment 29. The method according to embodiment 28 wherein the mitebody fraction in step a) or the combined body fractions in step b)comprises up to 20% v/v mite parts (e.g. legs), faecal particles andmite medium.

Embodiment 30. The method according to embodiment 29 wherein the mitefaecal fraction in step a) or combined faecal fractions in step b)comprises up to 20% v/v mite parts (e.g. legs) and mite medium.

Embodiment 31. The method according to any of embodiments 18-30 whereinthe fraction(s) of mite bodies and the fraction(s) of mite faecalparticles in step a) of embodiment 18 is isolated from mite culture(s)by sieving of the Der p and/or Der f mite culture(s) to produce a mitebody fraction b) and a mite faecal particles fraction d).

Embodiment 32. The method according to embodiment 31 wherein thefraction(s) of mite bodies and the fraction(s) of mite faecal particlesis obtained by sieving of the Der p and/or Der f mite culture(s) toproduce four fractions:

a) Large medium particles >350 μm

b) Mite body fraction 350-90 μm

c) Faecal agglomerates and mite parts 90-50 μm prepared

d) Faecal particles <50 μm

fraction a) and c) are discarded, fraction d) is used directly forextraction and fraction b) is subjected to further purification.

Embodiment 33. A method for the isolation and purification of mitebodies comprising the steps:

i) Isolating mite body fraction(s) from Der p or Der f mite culture(s)and

ii) Subjecting one or more Der p mite body fraction(s), or one or moreDer f mite body fraction(s), to density centrifugation in a first liquidmedium having iia) a higher density than the mite bodies and a lowerdensity than the culture media particles and iib) do not extract group 1and group 2 allergen form the mite bodies; and

iii) Collecting the mite bodies from the surface of said first liquidmedium

and optionally repeating step ii) and iii) until the desired purity ofthe mite bodies have been achieved, optionally followed by

iv) Washing of the mite bodies to remove said first liquid with a secondliquid that does not extract group 1 and group 2 allergen from the mitebodies.

Embodiment 34. The method according to any of embodiments 31-32 whereinmite body fraction b) is:

ii) Subjected to density centrifugation in a first liquid medium havingiia) a higher density than the mite bodies and a lower density than theculture media particles and iib) do not extract group 1 and group 2allergen form the mite bodies; followed by

iii) Collection of the mite bodies from the surface of said first liquidmedium and optionally repeating step ii) and iii) until the desiredpurity of the mite bodies have been achieved, optionally followed by

iv) washing of the mite bodies to remove said first liquid with a secondliquid that does not extract group 1 and group 2 allergen from the mitebodies.

Embodiment 35. The method according to any of embodiments 33 or 34wherein the first and second liquids are anhydrous, preferablypharmaceutically acceptable liquids.

Embodiment 36. A method according to embodiment 35 wherein the firstliquid is anhydrous glycerol.

Embodiment 37. A method according to embodiment 36 wherein the secondliquid is anhydrous ethanol.

Embodiment 38. The method according to embodiment 33-37 wherein step ii)and iii) is repeated once.

Embodiment 39. The method of any of embodiments 33-37 wherein step ii)and iii) are repeated until the mite body fraction obtained comprises atleast 70% v/v mite bodies, more preferred more than 75% v/v mite bodies,more preferred more than 80% v/v mite bodies, preferably more than 85%v/v mite bodies, preferably more than 90% v/v mite bodies, preferredmore than 95% v/v mite bodies and most preferred more than 98% v/v mitebodies.

Embodiment 40. The method of embodiment 31-33 and 38-39 whereinpurification of the mite body fraction is continued until the mite bodyfraction contain below 10% v/v culture media particles.

Embodiment 41. The method of embodiment 40 wherein the purification ofthe mite body is continued until less than 20% v/v mite parts, faecalparticles and mite medium is present.

Embodiment 42. A mite body fraction comprising more than 70% v/v mitebodies, more preferred more than 75% v/v mite bodies, more preferredmore than 80% v/v mite bodies, preferably more than 85% v/v mite bodies,preferably more than 90% v/v mite bodies, preferred more than 95% v/vmite bodies and most preferred more than 98% v/v mite bodies.

Embodiment 43. A mite body fraction according to embodiment 42containing below 10% v/v culture media particles.

Embodiment 44. A mite body fraction according to embodiment 43comprising less than 20% v/v mite parts, faecal particles and mitemedium.

Embodiment 45. The method of any of embodiments 18-30 wherein fractionsof Der f mite bodies, fractions of Der p mite bodies, fraction(s) of Derp faecal particles and/or fractions of Der f faecal particles in step 18a) is isolated by a sieving process comprising:

1) Sieving Der f or Der p mite cultures in a vibrating sieve shaker witha tower of a 300, a 150 and a 80 μm mesh sieve followed by isolation ofa mite body rich fraction between 150 μm and 300 μm and a mite faecalparticles rich fraction below 80 μm, followed by

2) Sieving the mite body rich fraction (150 μm and 300 μm) in a sieveshaker with a horizontally and circular movement and with a tower of200, 300 and/or 400 μm mesh sieves and isolating body fraction(s)captured by the 200, 300 and/or 400 μm mesh sieves, followed by

3) Sieving of the body rich fraction in a sieve shaker having a 3Dthrowing motion and 300, 200, 150 and 100 mesh sieve to produce a mitebody fraction between 150 and 300 μm mesh and a mite faecal particlesextract below 100 μm; and

4) Optionally combining the facal particles fraction below 80 μm fromthe vibrating sieve and the faecal particles fraction below 100 μm fromthe sieve with the 3D throwing motion and subjecting the combined faecalfraction to sieving in the sieve with the 3D throwing motion and a towerof 100, 80, 50 μm mesh sieves and isolating the faecal particles richfraction blow 50 μm.

Embodiment 46. The method according to any of embodiments 18-41, 45wherein the mixture of extracts (I) obtained is formulated as acompressed or non compressed sublingual tablet(s) comprising the extract(I) or allergoids prepared from extract (I) in a solid form, a liquidsublingual product or a liquid product for injection comprising theextract (I) or allergoids prepared from extract (I), preferably thepharmaceutical product is a fast dissolving sublingual tablet whichcomprises an extract (I) in solid form.

Experimental Section

EXAMPLE 1

1.0 Cultivation of Mites

Der p and Der f mites are cultivated separately on a suitable mediumwith medium particles which are of sufficient size and integrity towithstand the rigors of downstream processing and be easily eliminatedthrough the primary screening process. The mite medium suitably containsa protein source, carbohydrates, minerals, oil, vitamins and apreservative.

Mites are killed by freezing at −20° C.±5° C. when the optimal growthhave been reach.

1.1 Purification of Mite Bodies and Mite Faecal Particles

The mite culture (Der p or Der f) is dried to moisture content below 15%and sieved to produce four fractions:

-   -   1) Large medium particles >350 μm    -   2) Mite body fraction 350-90 μm    -   3) Faecal agglomerates and mite parts 90-50 μm    -   4) Faecal particles <50 μm

An automated sieving unit is used to screen the dried mite culture. Thesiever uses rotation at set angles and speed to obtain fractions. Thescreen that produces fractions 1, 2 and 3 above can be de-blinded duringsieving. The fines (fraction 4) are collected in the bottom pan of thesieving unit.

The large medium particles in fraction 1 and the faecal agglomerates andmite parts in fraction 3 are discarded.

The quality of the faecal particles in fraction 4 is tested (to assurethat the fraction contains mostly faecal particles and meets releasespecifications) and it is decided whether the fraction is suitable forextraction of Der p or Der f allergens.

The v/v % of whole mite bodies, mite body parts, faecal particles, mitemedia and contaminants are measured by microscopy of samples, see page13-14.

Several mite faecal fractions may be combined before extraction.

1.2.1 Purification of Mite Bodies Fraction 2 by Centrifugation withGlycerol

Centrifugation of fraction 2 is carried out in a (Beckman J6MIcentrifuge).

Not more than 900 grams of fraction 2 is slowly added to USP anhydrousglycerine (99.0-101.0% pure) in an amount that is five times the weightof fraction 2.

The mite culture mixed with anhydrous glycerine is poured intocentrifugation cups so that all cups contain the same volume. The cupsare placed in the rotor and the cubs are centrifuged at 3000 rpm for 7minutes at −5 ° C. The mite body plug formed is removed from the surfaceof the anhydrous glycerine and weighed. The mite bodies are mixed withan amount of anhydrous glycerine 3 times the weight of the mite bodyfraction and centrifugation is repeated.

The mite body plug is carefully removed from the surface of theanhydrous glycerine and weighed. The weight of the mite bodies ismultiplied by 2.5 to determine the minimum amount by weight of anhydrousethanol needed to form a slurry. The mite body and ethanol slurry ismixed for 5 minutes in a mixing vessel equipped with a low-shear blade.The slurry is poured onto two 106 micron sieves with a collection acollection pan.

Scrape the mite bodies from the sieves and record the weight of themites. Repeat the last purification step with ethanol one more time anddry the mite bodies using vacuum or are allowed to dry on the 106 sievein a fume hood.

The v/v % of whole mite bodies, mite body parts, faecal particles, mitemedia and contaminants are measured by microscopy of samples, see page13-14. The mite body fraction achieved typically comprises 98-99 vol/vol% whole mite bodies.

1.2.2

Mite Bodies from Four Different Lots are Subjected to Purification asGenerally Outlined Above in Example 1.2.1

In short, a centrifugation slurry was formed from mite culture fractionand anhydrous glycerine either 4, 5 or 6 times (weight ratio) and pouredinto centrifugation cups (up to 6 cups at the per run) which werecentrifuged once or twice at 2500 rpm or 3500 rpm, where after the mitebody plug were removed and washed 3 time with ethanol. The v/v % ofwhole mite bodies, mite body parts, faecal particles, mite media andcontaminants were measured by microscopy of samples see example 1.4.

The results show a yield increase when centrifugation once in each ofthe four D. pteronyssinus lots processed, with the increase ranging from72-156% with the average being 102%. For mites of the species D. farinaethe % increase ranged from 83-111% with the average being 93.5%.

The purity levels of the resulting body fractions obtained were allabove 97%, with faecal partical contents of below 2% medium contents of2% or below. The group 1 to group 2 ratio were below 1.

Mite body fractions from different mite cultures may be mixed.

1.3 Purification of Mite Bodies Fraction 2 by Centrifugation withSaturated NaCl

Mite body fraction 2 are mixed with saturated NaCl (in an amount that isfive times the weight of fraction 2) to a slurry. The slurry iscentrifuged at 3.000 RPM in 7 min (15° C. in a Sorvall centrifuge). Mitebody plug is carefully removed from the surface and resuspended in anamount NaCl that is five times the weight and the centrifugation isrepeated. The v/v % of whole mite bodies, mite body parts, faecalparticles, mite media and contaminants are measured by microscopy ofsamples, see page 13-14.

The mite body fraction achieved typically comprises 98-99 vol/vol %whole mite bodies.

TABLE 1b # 1 and 2 are saturated salt water 1 spin, # 3 and 4 aresaturated salt water 2 spin Volumetric Percent Composition Sample IDBodies Faecals Medium 1 98.200 1.180 0.620 2 98.100 1.230 0.660 3 99.8900.110 0.000 4 99.860 0.140 0.000 Table 1b

EXAMPLE 2

2.0 Purification of Mite Bodies and Mite Faecal Particles

Place frozen culture on trays inside the purification room. Make surethe culture is spread in an even layer on the trays and let the culturethaw and dry at ambient conditions for 4-14 days.

The culture is then fluidized to dry it and to separate it in 2fractions, see FIG. 1. An air flow is applied connecting a vacuum pumpin one side and an air inlet in the other.

The glass containers in FIG. 1 are named from left to right “A” with thewhole mite culture, “B” is rich in body particles and “C” is faecalparticles.

The two fractions (B) and (C) are isolated and subjected to sieving.

2.1 Purification of Mite Bodies

2.1.1 Big Sieve

Fractions B are added to a continuous charge industrial vibratory FiltraFT 500 sieve shaker. The sieve has a tower of three meshes: 300, 150,and 80 μm and work at 1500 rpm.

The material above 300 μm is mainly food particles and is discarded.

The material below 80 μm is further purified in small sieve to isolatefaecal fraction.

The material below 300 μm and above 150 μm was isolated as a body richfraction and sieved in the medium sieve below.

2.1.2 Medium Sieve

Mites have a form which is not regular and can reach 400 μm in onedimension but are usually smaller in dry form. Mites have legs and hairsand when a horizontal circular motion is used during sieving the mitebodies are oriented horizontally forcing the formation tangles. Only afew disorientated particles or smaller particles not forming part of thetangle enter the mesh and the sieve is not blocked so quickly.

The sieve used is a Retsch AS 400 sieve shaker with a tower of 400, 300and 200 μm meshes. A horizontal circular sieving mode is used and thesieving process takes advantage of the formation of tangles of miteshaving a size above 200, 300 and/or 400 μm.

The material from big sieve is divided in 150-200 g portions and addedto the second (medium sieve).

The sieve work at a speed between 210 to 240 rpm and each batch aresieved for 1-2 minutes.

Fractions above 200, and 300 μm are reprocessed 2-6 times.

The content of mite bodies captured on the 200, 300 and/or 400 μm meshsieves is determined by microscopic inspection of the fractions and thebody rich fraction is isolated an subjected to further sieving asdescribed below.

2.1.3 Small Sieve

The body rich fraction recovered from the Medium sieve is then subjectedto sieving in a small sieve (Retsch AS 200) using a 3-D throwing motion.The mean amount of body rich fraction loaded into the sieve is 60 g andA tower of 300, 200, 150 and 100 mesh is used.

The speed is set to 240-260 rpm, the amplitude is set to 30 seconds andeach load is sieved for 5-15 minutes. Each load is reprocessed 1-6times.

In process controls: During the sieving process purity of the fractionis tested using a microscope.

The fraction below 100 μm was isolated as a feacal particles fractionwhich was purified as described below.

The fractions above 100 μm and 150 μm are isolated as mite bodyfractions.

2.2 Purification of Faecal Fraction

The faecal rich fractions obtained in previous steps (fraction C ofpre-treatment step, fraction <80 μm of big sieve-shaker and fraction<100 μm of small sieve-shaker) are combined.

The small sieve-shaker (Retsch AS 200) is used with a tower of 100, 80and 50 μm mesh and the operation conditions were the same as mentionedabove for this sieve.

The fraction below 50 μm is isolated as the faecal fraction.

In process controls: During the sieving process purity grade of thefractions is estimated using a microscope.

EXAMPLE 3

3.0 Extraction Mite Bodies

Mite bodies were suspended using a high shear mixer (20 min). Extractionwas carried out in a buffer containing 0.012 M NaHCO₃, 0.154 M NaCl, ata pH of 7.5 for 3 hours at a temperature of 8° C. The mite body tobuffer were 1+10 (w/w).

3.0.1 Separation

The remaining insoluble source material is separated from the extractsolution by centrifugation.

After centrifugation the resulting centrifugate is decanted from thesolid fraction and clarified through a filter into a filtrate solution.After filtration of the allergen extract, the filter is washed withextraction buffer and pooled to increase yield.

3.0.2 Ultrafiltration

The ultrafiltration is performed in three steps: Concentration step,Diafiltration step and dry matter adjustment step. Firstly, the filtratesolution is concentrated by removing water and other small molecules bytangential flow filtration. Secondly, small molecules and salts areremoved from the allergen extract solution by diafiltration againstpurified water. Thirdly, the dry matter (DM) concentration of theallergenic extract is adjusted by further concentration or addition ofpurified water. During ultrafiltration the temperature andtrans-membrane pressure (TMP) are monitored and kept within theacceptance criteria.

In process control:

Temperature: 3-15° C.

Trans Membrane Pressure: 3 bar

DM concentration: 40-60 mg/ml

3.0.3 Clarification Filtration

The allergen extract solution is clarified through a 0.2 μm filter.

3.04 Stabilization

After filtration the extract is dispensed as free-falling droplets intoa container filled with liquid nitrogen where the droplets freezeimmediately. The liquid nitrogen is then allowed to evaporate. Duringthe stabilization process the feed flow (product flow) is controlled tobe within the acceptance criteria.

In process control: Product flow: 8-12 ml/min/dispensing hole.

3.1 Faecal Particles

The mite faecal particles are suspended using a high shear mixer (20min). Extraction was carried out in a buffer containing 0.012 M NaHCO₃,0.154 M NaCl, at a pH of 7.5 for 1 hour at a temperature of 8° C. Themite body to buffer ratio is 1+10 (w/w).

3.1.1 Separation

After extraction the insoluble source material is separated from theallergen solution by a diafiltration step. The diafiltration isperformed using a vibrating membrane filtration system (TFF technique).The allergen extract passes through the membrane as permeate, while theinsoluble source material will be retained by the membrane. During thediafiltration process a pre-defined volume of purified water is addedcontinuously to the recirculating extract solution at the same rate asthe allergen extract (permeate) is collected from the other side of themembrane.

In process control:

Permeate flux: 18-22 liter/m2/hour

Temperature: 4-12° C.

3.1.2 Ultrafiltration

The ultrafiltration is performed in three steps: Concentration step,Diafiltration step and Dry matter adjustment step. Firstly, the filtratesolution is concentrated by removing water and other small molecule bytangential flow filtration. Secondly, small molecules and salts areremoved from the allergen extract solution by diafiltration againstpurified water. Thirdly, the dry matter (DM) concentration of theallergenic solution is adjusted by further concentration or addition ofpurified water. During ultrafiltration the tempera-ture and transmembrane pressure (TMP) are monitored as in-process controls and keptwithin the acceptance criteria.

In process control:

Temperature: 3-15° C.

Trans membrane pressure: 3 bar

DM concentration: 40-60 mg/ml

3.1.3 Clarification Filtration

The allergenic solution is clarified through a 0.2μm filter.

3.1.4 Stabilization

After filtration the allergenic extract is dispensed as free-fallingdroplets into a container filled with liquid nitrogen where the dropletsfreeze immediately. The liquid nitrogen is then allowed to evaporate.During the stabilization process the feed flow (product flow) iscontrolled to be within in acceptance criteria.

In process control:

Product flow: 8-12 ml/min/dispensing hole

EXAMPLE 4

The content of group 1 and group 2 allergen is measured by MS (massspectrometry as described in WO 2007/031080 using the followingcalibration standard peptides:

SEQ ID NO: 1 TNACSINGNAPAEIDLR (Der p 1), SEQ ID NO: 2NSWDTTWGDSGYGYFQAGNNLMMIEQYPYVVIM (Der f 1), SEQ ID NO: 3VLVPGCHGSEPCIIHR (Der p 2). SEQ ID NO: 4 GKPFTLEALFDANQNTK (Der f 2),and SEQ ID NO: 5 GIEYIQQNGVVEER (Der f 1)

EXAMPLE 5

5.0 Mixing of Extracts

Frozen balls of mite body extract were added frozen balls of faecalparticles extract to achieve the desired ratio of group 1 and group 2allergen.

The determination of which batches of frozen balls to be used and thenumber of frozen balls to be used is an iterative process.

The mixture with the desired ratio is created by calculating the amountby weight of group 1 allergen that will result from mixing of a numberof balls from at least one batch of mite body extracts and at least onebatch of mite faecal particles. The amount by weight of group 1 allergenis calculated as the amount by weight of any batch multiplied with theconcentration of group 1 allergen in said batch. The amount by weight ofgroup 2 allergen resulting from mixing the same batches is calculated inthe same way.

More batches or parts of batches are added or removed from thecalculation as described on page 27 above until the desired ratio isachieved.

EXAMPLE

TABLE 2 Name Major (m_(F1), Production Major allergen allergen Extractm_(B1), m_(F2), batch group 1 group 2 (IM) m_(B2), etc.) No. [μg grp ·1/ml IM] [μg/ml IM] Body m_(B1) IMPTE2001-B 991 1223 Faecal m_(F1)IMPTE2001-F 2393 792

The amount of extracts from each batch is calculated to be 140 gram mf1and 966 gram mb1

Major allergen ratio=1=Total group 1/Total group2=mf1×Cf1grp1+mb1×Cb1grp1/mf1×Cf1grp2+mb1×Cb1grp2=2393.140+991.966/792.140+1223.966=1

The frozen balls required are thereafter thawed and mixed and optionallyfrozen as balls again. Thawed mixtures may be used in the formulationprocess.

EXAMPLE 6

The group 1 and group 2 allergens are determined as described in example5.

Batch FAR1 FAR2 FAR3 FAR4 FAR5 FAR5 FAR6 FAR7 CV (in %)* Ratio (Der1/Der 2) 0.96 0.97 1.00 0.95 0.89 0.88 0.95 0.95 6 Batch PTE1 PTE2 PTE3PTE4 PTE5 PTE6 PTE7 PTE7 CV (In %)* Ratio (Der 1/Der 2) 1.04 1.17 0.941.01 1.00 1.03 1.10 1.01 6.5 *CV = relative standard deviation(SD/mean), with 95% CI

EXAMPLE 7

6.1 Preparation of a Fast Dissolving Pharmaceutical CompositionContaining an Extract

Composition of house dust mite tablet before sublimation of water duringfreeze-drying:

TABLE 3 Weight Ratio of group 1 to Name of Ingredient group 2 allergenFunction Active Ingredient Der p mite body extract 1:1:1:1 ActiveIngredient Der p faecal particles extract Der f mite body extract Der ffaecal particles extract Other Ingredients Gelatin (Fish) 15 mg Formerof freeze-dried tablet structure Mannitol 12.7 mg Former of freeze-driedtablet structure Sodium Hydroxide to pH 7.5 qs pH adjustment PurifiedWater qs to 250 mg Vehicle Total Wet Dosing Weight 250 mg —

Preparation of the Tablet:

Gelatin, mannitol and purified water are mixed and the resulting premixis heated to appropriate temperature. Following cooling, the pH of thesolution is adjusted using sodium hydroxide solution (˜6% w/w). The drugsubstances are added to the premix as frozen droplets. The pH of the mixis tested and adjusted, if necessary. Additional amounts of purifiedwater required to complete the formulation is calculated using themanufacturing formula and transferred to the mix solution. The solutionis dosed into pre-formed blister trays. After dosing, the filled blisterpacks are passed through a liquid nitrogen freeze tunnel. All frozenproducts are immediately placed in a refrigerated cabinet for frozenstorage prior to freeze-drying. The units are freeze-dried and stored ina dry storage cabinet in a controlled humidity environment untilsealing. The freeze-dried units are then sealed with foil.

All patents, patent applications and non-patent literature cited hereinare hereby incorporated by reference.

1. A method for the manufacture of a mite allergen extract comprisingDer p 1 and Der p 2 allergen, which method comprises the followingsteps: a) isolating fraction(s) of Der p mite bodies from cultures ofDer p mites, wherein said fraction(s) comprises more than 70 percent v/vDer p mite bodies; b) optionally combining several of said fraction(s)of Der p mite bodies; and c) extracting allergens from said Der p mitebody fraction(s) obtained in step a) or a step b) as above to obtainsaid mite allergen extract, wherein step a) comprises the followingsteps: i) isolating mite body fraction(s) from Der p mite culture(s);ii) subjecting one or more Der p mite body fraction(s) to densitycentrifugation in a first liquid medium having a higher density than themite bodies and a lower density than the culture media particles; andiii) collecting the mite bodies from said first liquid medium andrepeating step ii) and iii) until the mite body fraction comprises atleast 70 percent v/v mite bodies, optionally followed by iv) washing ofthe mite bodies to remove said first liquid with a second liquid.